Handbook of Radiopharmaceuticals

Since N, is so large as to remain essentially constant during irradiation, the solution to this differential equation is

The activity of the product Ap at the end of bombardment (EOB) is XNp, and after substitution we obtain the radionuclide production equation

where A = activity in disintegrations per second, dps Nt = number of target atoms = W/M x F x Avog [W = sample weight in grams, M = atomic weight in grams/mole F = isotopic abundance, Avog = Avogadro's number; 6.023X1023 atoms per mole] cp = neutron flux in neutrons. cm–2. sec ' o = cross-section in cm2 A. = decay constant of product = ln(2)/t1/2 t = irradiation time Notice that at long irradiation times where Xt»l (or tirrad » t ./, of the product) the factor (1-e '**) approaches 1 . This signifies that the rate of product formation and rate of product decay are equal, and the reaction is said to be at "saturation". The term (l-e"^) is also known as the saturation factor. Thus longer irradiation time does not

REACTOR PRODUCTION OF RADIONUCLIDES

93

produce more radioactivity. In practice, irradiations longer than one half-life ("half saturation") are relatively inefficient since doubling the irradiation by another half-life only increases radioactivity from 50% to 75% of saturation. As an example of the use of the production equation, consider a 24 hour irradiation of 1 mg of natural rhenium (37.4% !85Re) in a thermal neutron flux of 2 x 1014 n cirflsec"1 to make 186Re (tI/2-3.718 d). The tabulated crosssection is 112 barns (Mughabghab et al. 1984). N, -( 0.00lg/186.21 gram per mole)(0.374 natural ab.)(6.023x!023atoms per mole) = I.21xl0 l8 atoms then the amount of 186Re radioactivity produced is A - (1.21 x 1018 atoms)(2 x 1014 n.cm'lsec^Xl 12 x 10"24 cm2)x [l-exp((-0.693/3.718 x 24 h) x 24 h)] = 4.6 x 109 atoms per second, or disintegrations per second (dps) = 124.3 mCi (1 mCi = 3.7xl04 dps) Corrections to this result would be needed if the sample were large, or the irradiation lengthened. For a massive target with high cross-section, there is sufficient absorption of neutrons in outer layers so that the effective neutron flux in the interior of the target is reduced (self-shielding). This effect is difficult to calculate and is usually measured. Also, 186Re itself can absorb a neutron and convert to 187Re. This process is called product burn-up. While some conversion of !85Re to 186Re can also be induced by higher energy neutrons, most comes from the thermal neutrons considered here because thermal neutron fluxes are usually much larger than for higher energy neutrons. The production equation can take one of two forms, depending upon whether the target is immersed in a bath of neutrons as in a nuclear reactor, or irradiated in a specific direction by a beam of particles from an accelerator. In the latter case, the target is usually larger than the diameter of the beam. The fraction of target irradiated is then important. A detailed discussion of the equations governing the production of radionuclides in a nuclear reactor can be found in Mirzadeh and Walsh (1988).

SOURCES OF NUCLEAR PARTICLES A. Nuclear fission reactor Although there are neutron sources based on natural radioactivity and accelerators, the most generally useful source of neutrons for radionuclide production is the research nuclear reactor. A reactor consists of an amount of fissionable material, either 235U (natural or enriched), 239Pu or 233U, assembled so that a controlled, self-sustaining chain reaction of neutron induced fissions is maintained. The fission of 235U produces on average 2.4 neutrons, two medium mass nuclei (fission products with average masses of 100 and 140 amu), and the release of about 200 MeV. Since fission is best induced with low energy neutrons, a moderator (usually water) is used to slow the emitted neutrons. Some neutrons are absorbed by the moderator and structural materials and some escape. A chain reaction can be sustained if at least one of the neutrons causes another 235U to fission. This condition is

94

HANDBOOK OF RADIOPHARMACEUTICALS

expressed as the multiplicity factor k and a reactor is "critical" at k=l. The 235U is usually embedded in high melting zirconium hydride contained in metal tubes bundled together into fuel elements. The arrangement of fuel elements is called the core. At steady state k is kept at 1, but sometimes k must be made larger than 1. This allows the neutron flux and the power level to be varied, and compensates for fuel use (burn up), build-up of neutron-absorbing fission products, and the deliberate introduction of samples that absorb neutrons. The quantity (k-1 )/k is called the reactivity, and reactors must be designed with excess reactivity. The reactivity and power level are determined by the position of control rods. These control rods are made of materials with large neutron capture cross-sections, such as boron, cadmium and hafnium. Lowering control rods into the core reduces the number of neutrons available for fission. Some specially designed reactors control reactivity by moving the fuel elements themselves. The most important fission product from the point of view of reactor operation is '35Xe. It has a half-life of 9.1 h and a neutron capture cross-section of 2.6 x 106 barns (b), the largest known. During steady state reactor operation the build up of 135Xe is slow. However, upon shutdown the amount of 135Xe initially increases rapidly due to grow-in from the decay of its parent, I35I (t ( /2 = 6.6h) and the decrease in (n,y) reactions which destroy it. This 135Xe "poisoning" peaks about lOh after shutdown, and causes a large drop in reactivity (as much as 30%). Since many reactors cannot overcome this much loss of reactivity, usually there is a period after shutdown during which the reactor cannot be restarted. A nuclear reactor produces neutrons with a spectrum of energies up to about 20 Me V; the most probable energy of neutrons emitted in the fission process is 1.5 MeV. A typical spectrum is shown in Figure 1. Neutrons are generally grouped into 3 categories, thermal neutrons (E,, < 0.4 ev), epithermal neutrons (0.4 eV < En < 100 keV), and fast neutrons (Ep > 100 keV). The energy spectrum of the lowest energy neutrons approximates a Maxwellian distribution similar to that of ordinary gas molecules in equilibrium at room temperature, thus the name thermal neutrons. The peak of this distribution is at 0.025eV. These neutrons are very efficient at producing nuclear reactions and are widely used commercially for radionuclide production. Epithermal (or resonance) neutrons are formed when fast neutrons are partially slowed down by collisions with moderator. The fast neutrons arise from the fission process directly. The use of reactors for power production is based on the large 200 MeV energy release per fission. Approximately 1 g/day of fissioned fuel produces 1 megawatt of reactor thermal power. Electrical output is only about one third the thermal power. Further discussion of reactor physics, safety and economics is beyond the scope of this chapter, but many references exist (Liverhaut, 1960; Glasstone & Sesonske, 1963; Cohen, 1977). Electrical power generation is generally incompatible with radionuclide production, so it is the research reactor which is of most interest here. These can take many different forms, but the vast majority use enriched 235U for fuel, with enrichment varying from 10% to greater than 90%. These typically thermalize the neutrons with moderators of water, D2O, or graphite. Power levels cover the range from 0.1 W (e.g., Aerojet General Corp. Teaching Reactor AGN-20) to 450 MW (Fast Flux Test Facility, Westinghouse Hanford Company). Useable

95

REACTOR PRODUCTION OF RADIONUCLIDES

13 10

sho12

CM

lio10 CD

10'

10"4

10"2

1

102

104

106

Neutron Energy (eV) Figure 1. Energy spectrum of reactor fission neutrons.

thermal neutron fluxes range from 5xl06 n cm"2 s'1 to ~2xl015 n cm-2 s"1. A summary of the maximum fluxes available at 9 of the principal research reactors in the United States is given in Table 1.

Most research reactors have the entire core immersed in water (or D2O) and are often called swimming pool reactors. The 5-8 meters of water function not only as a moderator but also for cooling and radiation shielding. Irradiation positions for samples typically exist between fuel rods (in-core locations) or in an annular region just inside a ring of low Z material with small neutron capture cross-section. This material (beryllium is often used) "reflects" neutrons back to the core and minimizes neutron losses. The reflector increases thermal neutron flux at irradiation stations and helps save fuel. The actual fluxes and neutron energies available for radionuclide production are a sensitive function of the detailed reactor design and vary with location within the reactor. Figure 2 shows a cross-section of the Oak Ridge National Laboratory High Flux Isotope Reactor and several sample irradiation tubes. A brief description of ORNL-HFIR and other major research reactors in U.S. can be found in Mirzadeh et al. (1992).

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HANDBOOK OF RADIOPHARMACEUTICALS

Table 1. Principal research reactors in the United States* Reactor/ Institution

Operating Power (MW)

Thermal

Neutron Flux Epi thermal

Fast Flux Test Facility**, Westinghouse Hanford Company

450

5.2xl014

6.6xl015

3.7xl014

Advanced Test Reactor, Idaho National Engineering Laboratory

250

4.5xl014

6.7xl014

4.1xl0 14

High Flux Isotope Reactor, Oak Ridge National Laboratory

85

5.3xl015

1.5xl015

5.8xl014

High Flux Beam Reactor**, Brookhaven National Laboratory

30

4.2xl014

1.3xl015

1.5xl014

Missouri University Research Reactor, University of Missouri

10

4.2xl014

1.6xl014

7.0xl013

Omega West Reactor**, Los Alamos National Laboratory

8

9.0xl013

5.0xl012

—

Georgia Institute of Technology Research Reactor

5

6.5xl013

1.7xl012

2.9xl0 12

Massachusetts Institute of Technology, Reactor

5

6.0xl013

S.OxlO13

Ixl0 1 3

Oregon State University Triga Reactor

1

l.OxlO 13

4.0x10"

—

Fast

'Data from Mirzadeh et al., 1992; ** recently shutdown

MAIN PRODUCTION APPROACHES WITH NEUTRONS Table 2 lists some radionuclides important to nuclear medicine produced at reactors. There are three general reaction types used: (a) neutron capture, [n,y];(b) neutron capture followed by decay; and (c) fission. The most widely used route is the [n,y] reaction with thermal neutrons. The advantage of this production process is its simplicity and high yield. It is straightforward in that in many cases elemental targets may be used and no chemical separation of target and product is required (or possible). Since cross-sections tend to be higher than most other reaction types, yields are generally good. The primary disadvantage of this reaction also relates to the fact that the radioactive product cannot be separated from the target. Thus stable atoms dilute the radioactive atoms and the specific activity will be much lower than that of "carrier-free" radionuclide. Radioimpuritie&niay arise from [n,y] reactions on other isotopic forms of the target or chemical impurities in the target. The use of isotopically enriched targets can minimize production of impurities and improve yield, but often at high cost.

97

Figure 2. Cross-section view of the core of the High Flux Isotope Reactor at Oak Ridge National Laboratory showing several tubes for introduction of samples for irradiation (courtesy, Oak Ridge National Laboratory).

Nevertheless, enriched targets are essential if the natural abundance of the target isotope is low and significant impurities are produced. In selected cases there is a technique that can be utilized to improve the specific activity of (n,y) produced radionuclides. This method is known as the Szilard Chalmers process (Szilard & Chalmers, 1934). The SzilardChalmers process depends upon the fact that, following neutron absorption, prompt gamma rays are emitted which may cause nuclear recoil and subsequent molecular bond disruption. This excitation sometimes leaves the resulting hot atom in a different chemical state from unreacted atoms and thus chemically separable. This separated fraction is relatively "enriched" in radioactive atoms and has higher specific activity than the rest of the target. The enrichment factor (E) is the ratio of specific activity of recoiled fraction to that of the bulk irradiated target. The target in this case must be a compound which is thermally stable and resistant to radiolytic decomposition from fast neutrons and gamma rays. For example, the Szilard-Chalmers effect has been utilized with tetraphenyl tin to improve the specific activity of 117mSn (Mausner et al., 1992).

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HANDBOOK OF RADIOPHARMACEUTICALS

Table 2. Reactor Produced Radioisotopes of Current Interest to Nuclear Medicine Reaction Cross-sections (barns)3 Radionuclide (tl/2)

Mode of Production

67

67

Cu(2.6 d)

"Mo(66 h)

117m

166

Sn(14.0d)

Ho(26.8\h)

0-th

Io

1.07±0.11)xl0–3

fast neutron sb

98

582.6±1.1 0.1310.06

14416 6.910.3

"6Sn[n,Y] 117 Sn[n,n']

(5.811 .2)xl0 3 (2.22±0.16)xlO''

(3.5010. 53)xlO' lc Epi-thermal neutrons0

165

Ho[n,y] Dy[n,y]l65Dy 165 Dy[n,y](6-)

61.2±1.1 (2.6510. 10)xl03 (3.6i0.3)xl03

650122 (3.4l0.2)xl02 (2.2l0.3)xl0 4

168

Yb[n,y]

(2.310. 17)xl0 3

(2.13l0.1)xl0 4

Lu[n,y] Yb[n,y]177Yb(B-, 1.9h)

(2.09i0.07)xl03 2.85

(1.09l0.04)xl03 6.3

Re[n,y]

11212

1717150

Zn[n,p]

235

U[n,f](6.07%) Mo[n,y]

164

169

Yb(32.0 d)

l77

Lu(6.7 d)

176 176

186

Re(90.64h)

185

Re(17.0h)

187

Re[n,y]

76.411.0

300120

W(69.4 d)

186

W[n,y]187W W(B".24h)[n,y]

37.9 14.5d

485115

Pt[n,y] Pt[n,n']

(4.2i0.8)xlO'2 (2.87i0.20)xl01

(5.26l0.79)xl02c Epi-thermal neutrons0

3.6610.19 98.710.1 (2.51i0.04)xl04

5414 1550128

188

188

187

194

195

199

Au(75.3 d)

198

Pt[n,y]199Pt(B-, 31 m) Au[n,y]198Au 198 Aufn,yl 197

ath: thermal neutron cross-section, I0: resonance integral All cross-sections from Mughabghab er al., 1981 , except as noted. b Mirzadeh et. al. (1986, 1992) c Mausnere/a/., 1985, Mirzadeh et. al. (1997b) d Mirzadeh (1998, unpublished) a

Another situation occurs as a result of an (n,y) reaction, in which an intermediate radionuclide decays to the product of interest. This process is used to make I25 I, for example, with the l24 Xe[n,y] 125 Xe—» I25 I reaction. The neutron capture product 125Xe beta decays to 125Xe with a 16.8h half-life. Because the final product can be chemically separated from the target, specific activity approaching the theoretical value for pure radionuclide is

REACTOR PRODUCTION OF RADIONUCLIDES

99

possible. Obviously, using high chemical purity targets and processing reagents is necessary to avoid introducing stable isotopes of the same element as the product. In the 125I example this means that both target and reagents should be free of stable iodine. It is also usually desirable to use an enriched target to minimize the coproduction of long lived or stable nuclides isotopes with the product. For example, irradiation of 124Xe containing a few percent of 126Xe (6.09% natural abundance) will result in production of stable I27I through the decay of I27Xe, and hence result in lower specific activity of 125I product In this case, since the target and product are chemically separable, it is possible and may be worthwhile to recover the enriched target material for reuse. A few useful radionuclides are produced with fast neutrons by [n,p] reactions (e.g., 35S, 47Sc, 64Cu, 67Cu), or by indirect reactions, such as that used for production of 18F. The [n,p] process shares many features with the [n,y] reaction followed by beta decay process described above, since the target and product are chemically separable. The indirect mechanism involves the use of an outgoing particle from an initial nuclear reaction to cause a second reaction in another target nucleus. Thus the neutron irradiation of 6Li creates a triton (3H) with sufficient energy to react with a neighboring 16O nucleus (in Li2CO3) to form 18F. The product is separable from the target(s). The typical neutron induced reaction types are shown schematically in Figure 3.

CD

£3

E

ZS

o o

"*— '

CL

Neutron Number (N) Figure 3. Change in proton and neutron number from most common neutron induced nuclear reactions.

Neutron induced fission of 2j5U creates fission products with atomic numbers ranging from Z = 30–66. The mass distribution of radioactive products from fission of 235U is shown in Figure 4. For the purpose of radionuclide production, targets containing about 25 grams of highly enriched 235U are irradiated and then chemically processed. Fuel rods also contain large amounts of 235U and its fission products, but are not processed for their radionuclide content. Given the large numbers of radionuclides created by the fission process, the chemical procedures to recover the radionuclides of interest may be quite involved. The most important medical

100

HANDBOOK OF RADIOPHARMACEUTICALS

60

70

80

90

100 110 120 130 140 150 160 170

Mass Number (A) Figure 4. Mass yield distribution from fission of 235U

radionuclides produced by fission are 131I, !33Xe and, of course, "Mo. The special role of "Mo and its daughter 99m Tc in nuclear medicine must be underscored. TARGETRY Upon determining the production reaction and irradiation conditions, one must select an appropriate target material. Whether for use in a reactor or accelerator (cyclotron), there are several general factors to be considered: 1. Physical and chemical form: Pure metals or elements are generally best. If not suitable due to the constraints below, favorable materials include alloys as well as simple compounds such as oxides, carbonates,

REACTOR PRODUCTION OF RADIONUCLIDES

101

halides, etc. This form must also be compatible with post-irradiation processing. Thus, easily dissolved compounds are sometimes preferable over metal targets. 2. Thermal properties: There can be substantial sample heating during irradiation. Incident particles impart energy and some nuclear reactions are exothermic. Although thermal neutrons do not transfer much energy, fast neutrons can. Additionally, some of the energy of prompt gammas from [n,y] reactions generated in the sample, fuel elements, reactor containment, and other nearby samples can be absorbed in the sample. The "gamma heating" is proportional to sample mass and can be substantial for samples in excess of a gram. Charged particles lose energy rapidly in a sample. The power in watts is equal to the energy deposited (MeV) multiplied by the beam current (in uA). For example, a thick target which degrades a proton beam 15 MeV at 100 uA, absorbs 1500 watts in a few square centimeters. Target cooling is thus essential in accelerators in order to prevent target overheating and possible destruction. In addition, the target material should have good thermal conductivity and a high melting point. For these reasons organic compounds and aqueous solutions cannot usually be used. 3. Chemical Stability: The target must not decompose at elevated temperature or evolve gases that would pressurize and rupture the containment capsule. Also, the target should not react with the encapsulating material. Finally, the sample must not be substantially decomposed by radiation damage. This is hard to predict a priori. This requirement generally excludes all organic material as a target for large scale production of isotopes 4. Purity: Targets with high radionuclide purity are often necessary to minimize radiocontaminants due to the activation of impurities. In compounds, the activation of all atoms must be considered. Sometimes isotopically enriched material is necessary to suppress competing reactions on other naturally occurring isotopes in the target. To achieve high specific activity, the amount of element of the final product in the target is crucial. For example, the Cu content of ZnO targets for 67Cu production must be <0.1 ppm. 5. Encapsulation: For safety and to prevent cross contamination, the target is almost always sealed in a primary container. For reactor irradiation at low neutron flux and short periods, small plastic tubes are useful as they do not activate appreciably and are inexpensive. At the higher neutron flux and longer irradiation time used for isotope production , samples are typically encapsulated in high purity quartz ampoules. These quartz ampoules are often placed in aluminum holders specific to the irradiation facility. The much higher level of energy dissipation in accelerators makes the construction of targets rather complicated (Weinrich, 1992). Further discussion of the vast array of accelerator target designs is discussed elsewhere in this volume. 6. Availability: The target material should be readily available commercially in high purity. 7. Target transport systems: Capability for the production of short-lived radioisotopes in a nuclear reactor is another important criteria to be considered. Access to the reactor irradiation facilities during reactor normal operation allows short irradiation periods, which are required for the production of many useful short-lived radioisotopes such as 47Sc, 64Cu, 67Cu, 105Rh, 109Pd, 153Sm, 166Ho and !99Au. These isotopes all have half-lives of about three days or less. Production of 99Mo can also be enhanced by on-line access to the targets.

The pneumatic tube facilities are typically used for irradiation of small samples in plastic or graphite capsules for accurate time intervals of a few seconds to 10 minutes or longer depending on the neutron or gamma heating of the facility. The pneumatic facilities are typically located in the reflector region of the reactor, and the capsule

102

HANDBOOK OF RADIOPHARMACEUTICALS

loading and unloading station is located in the experiment room of the neutron activation laboratory. The capsules are inserted into the reactor and returned to the laboratory using compressed air. The on-line access to the reactor irradiation facilities can be accomplished pneumatically, hydraulically and by the use of vertical thimbles. A hydraulic "rabbit" permits for insertion and removal of samples while the reactor is operating. The water pressure-drops that exist in the primary coolant system are typically utilized as the driving forces for moving the capsules through the system. Normally, the heat flux at the surface of the capsule, due to neutron and/or gamma heating of the capsule and its contents, is limited. For the hydraulic tube facility at ORNL HFIR, this limit is of the order of 2x102 kWm 2 per capsule. In addition, the neutron poison content of the facility load is limited such that the reactor is not subjected to a significant reactivity change during the insertion or removal of capsules. Thimble mechanisms allows access to the reactor simply by attaching the irradiation capsule to the end of long aluminum tubing and lowering the capsule into the reactor and to withdraw it after the irradiation. The thimbles are fitted with a set of reentrant tubes connected to a separate cooling system which circulates a flow of ten or more liter/min downward through the center and upward through the outer annular region of each tube. Samples are placed in aluminum capsules which are immersed in the cooling water, or thimble tubing can be used to vent the capsule, to fill it with gas for heat transfer, and to bring out a pair of thermocouple leads, if desired.

CHEMICAL PROCESSING Except for [n,y] reactions, most radionuclides require chemical separation from the target material and induced radioactive byproducts. Generally one or more of conventional techniques, such as chromatography, solvent extraction, distillation and precipitation are used. Other techniques include electrolysis and electro-deposition, and separations based on sublimation. These processing techniques are adapted to the unique requirements of radiochemistry - the hazards of radiation exposure and contamination, rapid separation times in order to minimize decay losses, and the separation of essentially massless (carrier-free) amounts of product from bulk target. A great deal of effort has gone into developing processes suitable for each application. 1. Solvent extraction: This technique involves the selective partitioning of particular chemical complexes between two immiscible solvent phases. Usually an aqueous solution of acid, base or salt and an organic solvent such as ketone, ether, amine or carbon tetrachloride are used. Its use in radiochemistry is partly due to the fact that partition coefficients are approximately independent of concentration down to tracer levels. It is also relatively simple and rapid to perform, can be extremely selective, and can be adapted to remote or automated operation. Extraction of a complex containing the radionuclide into the organic phase is often followed by evaporation or a back extraction into an appropriate aqueous phase. For example, extraction of Ga (III) and In (III) chlorides into isopropyl ether followed by evaporation of the ether or back extraction is a commonly used approach that has been adapted for 67Ga and '"in purification (Brown, 1971.1972). The variables to be controlled are pH, relative phase volumes, salt concentration and mixing time. 2. Chromatography: This method is one of the most powerful and widely used for radiochemical separations, especially ion exchange. It is related to solvent extraction in that it depends upon the differential distribution of a species between two phases, except that in chromatography the phases move relative to one

REACTOR PRODUCTION OF RADIONUCLIDES

103

another. In ion exchange the distribution of an element between a solution (mobile phase) and stationary resin (usually packed in a column) depends on the ionic form, the solute concentration, and the functional group on the resin. Cation exchangers such as Dowex 50 have sulfonic acid groups, while in anion exchangers such as Dowex 1 the functional group is quaternary amine groups. Once a resin type is chosen, the variables to be controlled are ionic concentration, column volume and diameter, flow rate, and eluant. With proper choice of conditions, ion exchange is very useful for separating carrier-free radionuclides from bulk target (mass ratio >108) having significantly lower affinity toward the resin. This method has been particularly successful in separating transition metals from each other and from the rare earth elements. A relevant example is the separation of 55Co from Fe target (Lagunas-Solar & Jungerman, 1979). It can be combined with solvent extraction for better separation factors, as was demonstrated in the case of 67Cu (Das Gupta etal., 1991). It is also readily adaptable to remote or automated operation. 3. Distillation: Some radiochemical separations can be affected by exploiting differences in volatility. For example, 131I can be separated from Te targets by dry distillation at high temperature (Evans and Stevenson, 1956) or from a dissolved target (Hupf, 1976). One can also distill the target away from the product, as was done to separate 32P from an elemental sulfur target (Evans & Stevenson, 1957; Mani & Majali, 1966). 4. Precipitation: In radiochemistry, precipitation plays less of a role than other separation applications. This is because of the carrier-free nature of the radionuclides - there is simply not enough mass to precipitate. Reversing the process, that is, precipitating the bulk target away from the product is sometimes used, but may still cause problems by carrying down the desired product by mass effects. Though occasionally useful for a first bulk separation or for low specific activity radionuclides, precipitation rarely has the desired selectivity for nuclear medicine requirements. Adsorption on walls of glassware and filter paper can be troublesome, and handling precipitates under remote conditions may be difficult. 5. Shielded facilities: The handling and processing of reactor produced radionuclides has to be carried out in specially designed radiochemistry laboratories with controlled ventilation and air conditioning, shielded remote handling facilities, and radioactive waste collection and storage tanks (Mausner, 1999). In most cases involving processing of reactor or cyclotron targets, radiation shielding for the chemist is required. This may be as simple as some stacked lead bricks inside a standard chemical fume hood. For pure beta emitters, small lucite disks mounted on tongs to shield the hands are all that is necessary. At higher levels of gamma radiation, a completely enclosed hot box or hot cell will be required. Hot cells usually have 4-8 inches of lead in the walls or several feet of concrete, lead glass windows, and master slave manipulators. 6. Current Requirements and Challenges: Specific activity is an important parameter since in many cases the availability of very high specific activity or carrier-free radioisotopes is required for biological applications. Specific activity is defined as the relative abundance of a radioactive isotope to the stable isotopes of the same element in a homogeneously mixed sample. Specific activity is usually expressed in terms of the disintegration rate per unit mass of the element (e.g. mCi/mg). One example of the importance of high specific activity is the radiolabeling of tumor-specific antibodies for both diagnostic and therapeutic applications where only very small amounts of the radiolabeled antibodies are administered to insure maximal uptake at the limited tumor cell surface antigen sites. Another example is the use of receptor mediated radiopharmaceudcals that are potentially very important for the clinical evaluation of neurological diseases. Since the population of neurotransmitter sites is very limited, high specific activity agents are required to evaluate site-specific uptake.

104

HANDBOOK OF RADIOPHARMACEUTICALS

Since the specific activity of a radioisotope produced by particle induced reactions is a direct function of the incident particle flux, an increase in the incident particle flux results in an absolute increase in the specific activity of the product. This relationship is linear for simple reactions and non-linear for complex reactions. It is important to note that the half-lives, production and destruction cross-sections, and irradiation time are equally important. Several important radioisotopes have long physical half-lives and low production cross-sections requiring long irradiation periods even in the highest neutron flux available. Examples in this category include tungsten-188 (half-life 69 days; parents for rhenium-188). In addition, the increased flux not only results in higher specific activity products but will result in conservation of the enriched target material. For example, increase of the neutron flux by a factor of two requires only about half of the enriched target material to produce the same amount of radioactive product. Considering the limited availability of the enriched materials, conservation is unavoidable. Sometimes a higher flux increases the potential capacity of the reactor by reaching the equilibrium conditions quicker. IMPORTANT SPECIFIC CASES A list of reactor-produced radioisotopes of current interest to nuclear medicine is given in Table 2 together with their mode of production and their corresponding nuclear cross-sections. From this list, the production and chemical processing of the following radioisotopes important in nuclear medicine, representing different types of nuclear reactions, will be discussed in some detail: -Molybdenum-99 produced by fission and neutron capture reactions -Tungsten-188 produced by double neutron capture -Lutetium-177 produced by both direct neutron capture, and indirect neutron capture followed by beta decay -Holmium-166 produced by both direct single neutron capture, and double neutron capture followed by beta decay -Tin-117m, and Platinum-195m produced by neutron inelastic scattering -Copper-67 produced by fast neutron induced reaction. Molybdenum-99. Radioisotope applications in diagnostic medicine have increased continuously in the past 30 years, and today one-third of the annual 40 to 50 million diagnostic procedures in the United States are performed with radioisotopes. The most widely used radioisotope in nuclear medicine is technetium-99m (99mTc, tin=6 h), the daughter of 66 h molybdenum-99 ("Mo). Technetium-99m (99mTc) is the principal radioisotope used in diagnostic nuclear medicine (70% of all radioisotope diagnostic procedures) with an estimated 10 million medical tests per year and 3-4 billion dollars cost to patients annually. Tc-99m decays by >99% IT (t|/2=6.0 h) to the ground state of 99Tc (t,/2=2.1xl05 y) with emission of 140.5 keV y-ray with intensity of 89%; about 10% of this y-ray also converts in the "Tc K shell. The B-decay of 99Mo feeds to "Tc and 99Tc, 87% and 13% of the time, respectively. In daily elution, specific activity of the "mTc approaches the theoretical value of 5 mCi/ug, due to the absence of a stable isotope of Tc in nature (Table 3). Over the past 40 years, several versions of 99Mo/99mTc generator have been developed, the most common being the original generator proposed by the Brookhaven group (Richards, 1966a, c; Subramanian, 1976). In this system, Mo is adsorbed on alumina, and separation of

REACTOR PRODUCTION OF RADIONUCLIDES

105

99m

Tc is achieved by saline elution. This chromatographic generator has the advantage of being portable and

simple to operate. It provides 99raTc in high efficiency (~70%) and with acceptable quality. Other 99Mo/99mTc generators in use include extraction-based and sublimation generators. A review of the latter can be found in Boyd (1982) and Mirzadeh and Knapp (1996).

Table 3. Nuclear Reactions for Production of 99,Mo 235 U[n,f]99Mo 99 • Produces high specific activity 99, Mo • •

Requires highly enriched 235U target Complex chemical processing

• •

Requires dedicated processing facility Generates high-level radioactive waste

8

Mo[n,y]99Mo Requires highly enriched 98Mo target Produces low specific-activity 99Mo Simple chemical processing Generates minimal waste

At the present time,99Mo is exclusively produced from fission of uranium-235 (235U), and in North America this isotope is solely available from Nordion, Canada. Due to the logistical difficulty associated with 99Mo production and distribution (i.e. short half-lives of 99Mo and 99mTc, complex chemical processing, status of reactors and processing facilities, and wide distribution of users) only 5 to 10% of the produced 99Mo typically reaches the users. Another important issue associated with the Mo production is the waste and environmental impact of fission-produced Mo. The handling and disposal of high-level radioactive waste containing 235U generated from production of 99Mo are serious issues, and as the result, no commercial production facility is currently operating in the U.S. Further, since 99Mo cannot be stockpiled, even short-term disturbances in labor situations and interruption of transport systems can have very serious consequences. There are several distinct long-term disadvantages for the routine production of 99Mo via fission. The fission route produces very high levels of radioactive waste including several radioactive gases, thus requiring dedicated facilities which are expensive to operate. Secondly, in the fission produced 99Mo, the target material is highly enriched 235U used in nuclear weapons, and thus requiring extensive security and safeguards. As a part of the international non-nuclear proliferation movement led by United States, there is concern about availability of highly enriched 235U in the near future. During chemical processing of irradiated 235U, there exists potential for catastrophic criticality accidents resulting in release of highly radioactive fission products. T

An important alternative non-fission route for production of Mo is via neutron capture 98Mo target — a process which does not present any of the drawbacks listed above. A comparison of the fission and neutron produced 99

Mo is given in Table 4. For fabrication of 99Mo/99mTc generators, the principal issue which differentiates fission-produced 99Mo from the direct neutron capture route is specific activity (SA). The commercial /99m. 99 manufacturers of•99, Mo/99m Tc generators require SA of 1-2 Ci/'mg. Fission-produced Mo has SA of ~1000 Ci/mg, whereas SA of the neutron-capture-produced99Mo, even in the highest available neutron flux (hydraulic

tube of the ORNL-HFIR), is approximately 50100 mCi/mg ~ a factor of 10-20 times lower than required for the traditional single stage generator system. In the most widely used generator, 99Mo is loaded onto a column of

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HANDBOOK OF RADIOPHARMACEUTICALS

alumina and 99mTc is eluted from column with normal saline. The required bolus volume for the quantitative elution of 99mTc obviously depends on the size of the column that in turn is inversely proportional to the specific activity of 99Mo. Thus lower SA of neutron-capture-produced the activity concentration of

99

Mo requires larger alumina column, and hence

99m

Tc (mCi /mL) would be lower.

Table 4. Fission-produced 99Mo -- Some General Information

6.1%, 142 Ci/g of 235U (t irr =7d at
EOB):

Achievable SA (at EOB):

4.8x102 Ci/mg of 99Mo 7.2x101 Ci/mg of Mo (tirr=7 d at cp n =7xl0 13 n.s '.cm 2) 1-2 Ci/mg 99.9999% 99.99% For each Ci of 99Mo, 100 Ci of other fission products are produced

Reactor Produced Tin-117m, Tin-119m, and Platinum-195m. Tin-117m is currently in Phase II/III clinical trials for bone palliation (Atkins etal. 1993, 1995). Platinum-195m, is an important tracer in evaluation of the pharmacokinetics of several antitumor drugs [e.g. cis-dicholorodiammineplatinum(II)] and for Auger electron (Willins & Sgouros, 1995; Mirzadeh & Packard, 1995; Hoeschele et al, 1982). Tin-119m is one of the major radionuclides used in Mossbauer spectroscopy, (Dickson & Berry, 1986). The production of metastable nuclei such as

17m

Sn,

195m

pt and 119m Sn via neutron radiative capture reactions are

characterized by small neutron cross-sections and, hence, low production rates. Metastable nuclei typically have excitation energies on the order of 100 keV and large differences in angular momentum from ground states (most metastable nuclei have high angular momentum). An alternative route for producing these types of metastable nuclei is through neutron inelastic scattering where the cross-section of the AZ[n,n']AmZ reaction is, in some cases, substantially higher than the cross-section for the (A-1 ) Z[n,y] Am Z route. As has been shown for the case of 117m Sn (Mausner et al., 1985), the magnitude of gain in the cross-section may compensate for the relatively lower fast neutron flux from a well-moderated fission spectrum. Note that the excitation energy of metastable nuclei will represent the threshold for inelastic scattering. Large research reactors, such as HFIR and HFBR, with significant epithermal and fast neutron fluxes are well suited for these types of reactions. A systematic study of the production of 117mSn, 1l9mSn and

195m

Pt in the hydraulic tube facility of the HFIR has

been reported (Mirzadeh et al., 1997b). In all three cases, the yields from the [n,n'] reactions were higher than those obtained from the [n.y] reactions. The relative gains in the specific activity of the unfiltered (no Cd filter)

REACTOR PRODUCTION OF RADIONUCLIDES

107

targets were 1.4, for 195mPt, 3,3 for 117m Sn and 4.4 for119mSn. The larger gain for 119m Sn could be attributed to the relatively lower excitation energy (89.5 keV) of this metastable nucleus. The excitation energies for 117mSn and 195m

Pt are 314.6 and 259.2 keV, respectively. Since the thresholds for these inelastic scattering reactions are well

above the cadmium cutoff, Cd filters did not have any effect on the yield of these reactions. The corresponding cross-sections for the inelastic neutron scattering reactions for the production of 117m Sn, 119mSn and

!95m

pt are

222+16, 68+12, and 287+120 mb, respectively. A value of 176+14 mb for the cross-section of 117 Sn[n,n'] 117m Sn reaction obtained at HFBR was reported earlier (Mausner et al, 1985). In the case of 116 Sn[n,y] l17m Sn reaction, the measured thermal neutron capture cross-section (ath) and resonance integral (I0)are 5.811.2 and350153 mb, respectively. For 118 Sn[n,y] 119m Snreaction,theo eff is 13.2+2.0mb. The measured thermal and resonance integral for this reaction are 7.4+2.0 nib and 144+22 mb, respectively. The measured ath and I0, for 194Pt[n,y]195mPt are 42+1 mb, (5.3+0.8)xl02 mb, respectively. As demonstrated for these three cases, in the determination of the neutron capture cross-sections contribution from inelastic neutron scattering to overall reactions are significant and appropriate corrections are required even when highly enriched target isotopes are used. Consequently, measured cross-sections for capture reactions are lower than reported values, The large-scale production yield of' 117mSn via inelastic scattering reactions were recently measured at the HFIR for a number of targets ranging in mass from 4 to 200 mg, enrichment of 87–92%. For one reactor cycle (~21 d) irradiation at the position 5 of the HT facility, the average yield for 87% enrichment is 7.9+0.8 mCi/mg of Sn (corresponding to 9.2 mCi/mg of U7Sn). The average yield at saturation is 12.911.2 mCi/mg of Sn. For a one-cycle irradiation and at the end of bombardment (EOB) the radionulidic impurities in 117mSn samples include 115 d 113Sn (0.10%), 2.7 d 122Sb (0.22%), 2.7 y 125Sb (0.03%) and 12.4 d 126Sb (0.03%). These values represent the average of nine runs using 84.23% enriched 117m Sn. In three runs with 92% enriched target, the impurities were 113Sn (0.03%), 122Sb (0.13%), 125Sb(0.05%) and 126Sb (0.01%). The more highly enriched target produces a purer product as expected. Antimony-125 is primarily produced from the beta decay of 9.5m 125mSn. This isotope can also capture a neutron to form 126Sb. The 122Sb is likely produced from neutron capture of stable 121

Sb formed from the beta decay of 121Sn. Two other potential contaminants are 27 h 121Sn (a pure beta emitter

and not detectable with y-ray spectroscopy), and 9.64 d 125Sn (also a parent of 125Sb). The level of 121Sn at 7 days post EOB is estimated at ~0.1%. No 125Sn was detected in any run and its level was estimated to be «0.01 %. The synthesis of the DTPA complex of i!7m Sn for bone pain palliation has been described (Srivastava et aL, 1998). Holmium-166. Holmium-166 (166Ho) is utilized in medical radiotherapeutic applications due to its physical properties which include high-energy beta radiation [E6|=1855 keV (51%), E62=1776 keV (48%), and E6av=666 KeV], a 26.4 h half-life and decay to a stable daughter. In addition, 166Ho has chemical characteristics suitable for protein labeling with bifunctional chelates. Holmium-166 also emits low intensity and low energy gamma rays (80.5 keV, 6%) which are suitable for imaging. Due to the absence of high energy gamma rays in its decay, 166

Ho may be used for outpatient therapy without significant external radiation to other individuals (Dadachova,

et al, 1995a, 1997; Smith et al, 1995).

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HANDBOOK OF RADIOPHARMACEUTICALS

Although 166Ho with moderate specific activity can be produced by the 165Ho[n,y]l66Ho reaction, its radionuclidic parent, 166Dy (t1/2=81.5 h), can serve as a source of high specific activity l66Ho. Dysprosium-166 is produced by double neutron capture reaction on 164Dy (Table 2). In certain applications, such as protein labeling, the use of a high specific activity radioisotope is essential. In addition, generator-produced 166Ho is free from 1200-y 166mHo which is unavoidably co-produced with 166Ho by the l65Ho[n,y ] reaction. For a four-day irradiation of 13.3 mg of natural Ho (monoisotopic) as Ho2O3 at the position 5 of HT-HFIR, the experimental yield of 166Ho is 96.5 Ci, in comparison to the theoretical yield of 75.7 Ci. Therefore, the specific activity of !66Ho at saturation at the end of bombardment would be ~10 Ci/mg of l65Ho. The experimental yields of 166Dy is 3.5 and 2.2 Ci/mg of 166Dy for 8 and 1 days of irradiation in positions 5 and 6 of HT, respectively. Although, the yield of the indirect route is lower than the direct route by a factor of 3, the specific activity of carrier-free 166Ho produced from beta decay of 166Dy would be much higher -- depending on the degree of separation between Dy and Ho. Because of the uniform chemistry exhibited throughout the lanthanide series, the separation factors between the adjacent members, however, are not very large. The reverse phase ion exchange chromatography technique has been used extensively for separation of various members of lanthanides, and the applicability of this technique for the separation of carrier-free l66Ho from milligram quantities of Dy has been shown (Dadachova et al., 1994). In reverse phase ion exchange chromatography, Ho and Dy are partitioned between the cation exchange resin (AG 50W or Aminex-A5) and the mobile phase containing the weakly complexing ligand a-HIBA at pH 4.3-4.6. As a consequence of "lanthanide contraction" and smaller ionic radii, the complex of a-HIBA with Ho has slightly higher thermodynamic stability than that with Dy, and the elution pattern is reversed with Ho being eluted first. The log 6 (overall stability constant) of Ho and Dy complexes with a-HIBA are 7.67 and 7.24 at 0.1 ionic strength, respectively (Dadachova et al., 1994). Under optimum conditions of [a-HIBA]=0.085 M, pH=4.27, T=25° C and flow rate of 0.8 mL/min, quantitative separation between Ho and Dy was achieved in a metal-free HPLC column containing Aminex-A5 resin operated at 1400 psi, with a separation factor of ~ 1000. Further separation of the purified 166Ho from a-HIBA was achieved with a small column of AG 50Wxl2 from 1 M HC1 solution followed by elution of the ionic Ho3* from column with 6 MHCL. Lutetium-177g. Lutetium-177 ( l77 Lu) is utilized in radioimmunotherapy when chelated to tumor-associated antibodies and has also been proposed as a radioisotope source in brachytherapy. Lutetium-177 decays with a half-life of 6.7 d with gamma rays of 113 keV (6.6%) and 208 keV (12%) suitable for deep-organ imaging. The average 8 energy of I77 Lu is 0.133 MeV and the average equilibrium dose rate constant for l77 Lu is estimated to be -0.5 g-rad/uCi-h. The rather longer half-life of l77Lu is better suited for slow-targeting antibodies, and it's lower equilibrium dose rate constant make 177Lu useful for radiotherapy of soft tissues. In addition, 177Lu has chemical characteristics suitable for protein labeling with bifunctional chelating agents such as the eight coordinated DTPA, DTPA derivatives or DOTA. Although Lu is the heaviest member of the lanthanides, the ionic radius of Lu3+ is expected to be comparable to that of Y3+ as the result of lanthanide contraction. In coordination number 6, the ionic radius of Lu3+ is 89.1 pm, ~4 pm smaller than that of Y*3. At 25° C and 0.1 M ionic strength, the

REACTOR PRODUCTION OF RADIONUCLIDES

109

equilibrium constant of Lu-DTPA complex (ML/M.L) is 2.51 xl022 M-1 in comparison with 1.12xl022 M-1 for the Y-DTPA complex (Martell & Smith, 1974). Due to rather large cross-sections, high specific activity 177Lu can be obtained directly by 176Lu[n,y] reaction (Table 2) . The natural abundance of 176Lu is, however, only 2.6%. The highest available in the United States is 73%. Alternatively,

176

Lu enrichment readily

177g

Lu can be obtained indirectly from beta decay of 176Yb

(t11/2=1.9 h, EBmax=400 keV), (Table 2). In this case, the 176Yb parent nuclei is produced in a fission nuclear reactor with neutron capture on 175Yb which has a natural abundance of 12.7%. Enriched 175Yb up to 96% is also available in the United States. It is clear that the indirect route yields higher specific activity preparations of 177g

Lu, if Lu can be separated efficiently from Yb target material. A similar separation, for carrier-free

166

Ho

177

from small quantities of Dy, was discussed in the preceding section. In addition, the indirect route produces Lu which is free from 160 d 177mLu. This impurity is unavoidably co-produced with 177Lu by the 176Lu[n,y] reaction. For a one-hour irradiation of two natural Lu targets, 4.8 and 9.1 mg as Lu2O3, at the position 4 of the HFIR hydraulic tube, the experimental yield of 177Lu was 66 mCi/mg of Lu, corresponding to a value of 2.5 Ci/mg of !76 Lu. The ratio of the I77mLu/177Lu in this case is 4.8xl0-3%. Under similar conditions, the yield from 43% enriched

l76

Lu targets (0.5-0.7 mg) were 1.1 Ci/mg of Lu and 2.6 Ci/mg of 176Lu with a 177mLu/177Lu ratio of

4.6x10 3%. These data indicate that a yield of ~80 Ci per mg of 176Lu can be expected within 4 days in the position 4 of the hydraulic tube. Obviously, the 177mLu/177Lu ratio will increase with the irradiation time. For a 4 day irradiation the fraction of 177mLu at EOB is expected to be less than 0.01%. The yield of 177Lu from the indirect route (from decay of I77Yb) is 2.0 mCi/mg of 176Yb for 1 h irradiation at the 5 position of the hydraulic tube. Although, the yield from indirect route is lower than the direct route by a factor of 1000, the specific activity of the 177Lu from both routes will be almost the same assuming carrier-free Lu can be separated from Yb in i part per thousand. Tungsten-188. Currently there is considerable interest in rhenium-188 (188Re) for various medical applications (see Knapp, et al., 1997 and references within). The convenient 16.9 hour half-life and 100% beta emission with high end-point energies (Eeav-764 keV) make l88Re an attractive candidate for radiotherapy of larger tumors . Rhenium-188 provides an ideal y-ray at 155 keV for imaging with an intensity of 15%. About 5% of this transition also converts at the electronic shell of the Os providing low range secondary electrons and X-rays. The average equilibrium dose rate constant for !88Re is 1.78 g-rad/uCi-h. Another major advantage is the availability of 188Re in the carrier-free state from a generator system with a shelf life of many months. The element Re is placed in Group Vllb of the periodic table under Mn and Tc. These three elements have numerous valance states and complex electrochemistry. Similar to Tc, the most stable oxidation state of Re is +7 perrhenate (ReO4~). In most complexes with ligands, however, Re assumes oxidation states of 4 or 5. Although there is some chemical resemblance of Re to that of Tc, there are significant chemical differences. The parent nuclide, 188W, is produced in a nuclear reactor with double neutron capture on highly-enriched 186W. The thin-target production yield of 188W as a function of irradiation time at a neutron flux of 2xl015 ns-1cm-2 (HFIR) is shown in Figure 5 (Mirzadeh et al., 1997a ). The cross-section for the production of 187W, the intermediate radionuclide, is 37.9 b (Mughabghab et al., 1984). The thermal neutron cross-section for

110

HANDBOOK OF RADIOPHARMACEUTICALS OANL-OWGMM4MS '

"

1

0

-

1

1

1

1

)

10

1

1

!

20 30 IRRADIATION TIME (d)

40

Figure 5. Thin target production yield of 188W as a function of irradiation duration at HFIR (neutron flux 2 x 1015 ns-1 cm-2).

!87

W[n,y]188W reaction is 14.5 b. The chemical form of the target can be either metallic or oxide. The main advantage of a metal target is a net increase in the yield per unit target, as a larger amount of W metal can be packed into an irradiation capsule. This is an important factor for the large scale production of 188W. The chemical processing of the neutron irradiated WO3 targets involves dissolution in excess of hot 1 M NaOH in the presence of H2O2 or NaOCl or both (Knapp et al., 1994). One approach to process metallic W is to take advantage of the reactivity of molecular oxygen toward metallic W powder at elevated temperatures, and subsequent dissolution of the WO3 as above. This approach has an additional benefit in that the technique can be used for simultaneous separation of W from Re and Os which form volatile oxides (Mirzadeh et al., 2000). W-188/Re-188 Generator System. Because of the chemical similarity to the "Mo —»"Tc pair, most efforts have focused on alumina-based generator systems where tungsten is retained on alumina in the form of tungsten oxide, tungstic acid, tungstates, isopolytungstates or phosphotungstates, and 188Re (formed from the beta decay of 188 W) is eluted with NaCl solutions (Callahan etal, 1989; Griffiths et al., 1984; Botros et al., 1986). Sorption of W-containing species was studied under various conditions including concentration and chemical form of tungsten, pH, and anionic nature as well as solvent quality, and the treatment and granularity of the alumina support(Botros et al., 1986; Kadinaero/., 1990). Alternatively, in a "Gel-Type" system 188W is co-precipitated

REACTOR PRODUCTION OF RADIONUCLIDES

111

with zirconium hydroxide to form a gel, which is then packed in a column, and 188Re is eluted with normal saline (Ehrhardt et al., 1987, 1990, 1992). Other systems developed over years include zirconium oxide column (Lewis, 1966), tungsten fluoride absorbed on Dowex 1 anion exchanger in the fluoride form (Balchot et al., 1969), and phosphotungstate on alumina (Mikheev et al., 1972). The adsorption functions of WO42 and ReO4 on alumina from HNO3, HC1, and NaCl data indicate that the highest distribution constant (Kd) of 104 for WO42 is obtained in 0.21 M NaCl, yet in the same solution the Kd of ReO4 is only 1.3 (Botros et al., 1986). An alumina-based 188W~*188Re generator was evaluated for !88Re yield and elution profile and !88W breakthrough using various reagents for a three-month period (Kamioki et al, 1994). The optimum concentration of cations for sharp elution of 188Re is ~0.05 M. Concentrations below this optimal value resulted in an increase in retention time as well as an increase in the FWHM of elution curve. While NH4+ and Naf ions had similar effects on the 188Re elution behavior, the NH4+ ions were found to be more effective than H+ ions of equal concentration. The yield of 188Re from the generator displayed a gradual decrease, from 80% to 64%, during the 3 month evaluation period. The breakthrough of 188W per elution was as high as 1 .2x 10 2 % but. decreased rapidly to 3.2xl0 -4 % within the first five elutions. In parallel studies, the adsorption dynamics of tungsten on alumina showed a sharp rise in the tungsten breakthrough at a W/A12O3 ratio of -120 mg/g, corresponding to a Kd of 8.4xl03 from nitrate solution at 0.05 M ionic strength (Dadachova et al,, 1995b). In continued studies, the surface interaction between WO42" ions and the alumina support in the 188W—l88Re/Al3O3 biomedical generator system was examined by the dynamic adsorption method, which employs the 187W tracer, and by FT-IR, Raman, 27A1 MAS NMR, and XPS spectroscopic methods. These studies demonstrated the complex physical and chemical nature of WO42 ion adsorption on alumina. The dynamic adsorption studies indicated that the adsorption process includes several stages, from initial monolayer formation to almost complete saturation of the alumina surface. FTTR, Raman and 27A1 MAS NMR data showed that adsorption of WO24 on the A12O3 surface in slightly acidic solutions proceeds via formation of thermodynamically stable complexes of tetrahedrally coordinated aluminum atoms and WO42 tetrahedrons. The conclusion on the nature of WO42 -Al2O3 complex in aqueous solutions was further supported by the results from XPS studies, where it was shown that Wcontaining species are strongly attached to the surface of alumina via W-O bonds (Dadachova et al., 1 995b). The required volume for quantitative elution of l88Re from a generator obviously depends on the size of the column which in turn is inversely proportional to the specific activity of 188W. The production of high specific activity 188W, however, is limited to only a few research nuclear reactors worldwide which have neutron fluxes of greater than 1015 n-s -1 cm -2 . To make use of low specific activity 188W that can be produced in nuclear reactors with lower neutron fluxes, tandem alumina/anion-exchange generator systems have been proposed (Kamioki, et al., 1994). In one approach, the proposed system was based on the observation that carrier-free 188Re was strongly retained in a small anion exchange column from dilute HNO3 and then eluted with strong HNO3 in a small volume (Kamioki et al., 1994). Copper-67. There continues to be considerable interest in exploring the potential use of 67Cu for therapeutic applications (Cole 1986; DeNardo etal., 1991; DeNardo etal., 1999; Roberts et al., 1989; Green et al., 1988; Deshapane et al., 1988; Roberts etal, 1987; Green etal., 1986; Maziere etal., 1983; Pastakia
112

HANDBOOK OF RADIOPHARMACEUTICALS

Copper-67 can be produced in an accelerator by Zn[p,xn] and spallation reactions, and in a reactor by 67Zn[n,p] reaction. The saturation yield of 67Cu from 67Zn[n,p] reaction at EOB is 112± 10 uCi/mg of67Zn at the 5 position of hydraulic tube of HFER. The production data from two reactors together with fast neutron fluxes available in the irradiation facilities are summarized in Table 5. Table 5. Reactor Production of Carrier-free

67

Cu

Reactor/ Position

Fast Neutron Flux (En>l MeV) (n. s"'.cm'2)

Sat. Yield of CuatEOB (uCi/mgof 67Zn)

Crosssection, (mb)

HFIR (85 MW) Hydraulic Tube

4.4xl014

110110

1.07±0.11a

HFBR (60 MW) In-core, VI 5 Core-edge, VI 1

3.0xl014 6.4xl013

90.2±9.5 14.411.6

1.23±0.13b 0.91±0.10b

67

(a) Mirzadeh and Knapp, 1992; (b) Mirzadeh et al, 1986

Techniques for separation of copper at trace concentrations from other metal ions include extraction [Handley (1963), Brown et al. (1969), Hasany (1980), Dasgupta (1991)], ion-exchange [O'Brien (1968), Neirinckx (1977), Barnes et al. (1982), Grant et al. (1982), Maziere et al. (1983) Bently et al. (1984), and Polak (1986)], dry distillation, Fritze (1964), and electrolytic separation [Maziere et al. (1983) Bentieyetal. (1984),and Mirzadeh et al. (1986). The application of the spontaneous electro-deposition technique for separation of carrier-free radioisotopes of copper from proton- or neutron- irradiated zinc targets was reported by Mirzadeh and Knapp (1992). In this process, electro deposition occurs without an external EMF. The absence of measurable current in this process eliminates the hydrogen over voltage and preserves the nobility of the Pt electrode. Consequently, the highest degree of separation of 67Cu+2 from interfering metal ions (i.e. ions of Co, Fe, Ni, etc.) was obtained. Separation factor of > Ixl0 7 was easily achieved from macro amounts (grams) of Zn with in 30 minutes. CONCLUSIONS In this chapter we have reviewed the general nuclear physics and nuclear chemistry relevant to producing radionuclides with neutrons in research nuclear reactors. Specific detail for several radionuclides of importance to nuclear medicine has been presented, each chosen to demonstrate a different technique. The variety of radionuclides, mostly neutron rich, which can be produced is large. Reactors are also critical to create the very large quantity of radioactivity that is often needed for clinical care. Thus the future need for reactor production of medical radionuclides is assured.

REACTOR PRODUCTION OF RADIONUCLIDES

113

REFERENCES Atkins HL, Mausner LF, Srivastava SC, Meinken GE, Strub RF, Cabahug CJ, Weber DA, Wong CT, Sacker DF, Madajewicz S, Park TI and Meek AG (1993) Biodistribution of Sn-117m(DTPA) for palliative therapy of painful osseous metastasises. J. Radiology, 186, 279-283. Atkins HL, Mausner LF, Srivastava SC, Meinken GE, Cabahug CJ and D'Alessandro TD (1995) Tin-117m(+4)DTPA for palliation of pain from osseous metastases. J. Nucl. Med., 38, 725–129. Balchot J, Herment J and Moussa A (1969) Un Generateur de Re-188 a Partir de W-188 Int. J. Appl. Radiat, hot., 20, 467–470. Barnes JW, Thomas KE, Bentley GE, Grant PM and Miller DA (1982) Irradiation and separation methods for 67 Cu. Los Alamos National Laboratory, Chemistry and Nuclear Chemistry Div. Progress Report LA9381-PR, 114–117. Bentley G and Taylor W (1984) Electrolytic isolation of Cu-67 from proton-irradiated zinc oxide. Fifth Int. Symposium Radiopharm. Chem., Tokyo, Japan, p.287; also see Rogers et al. (1984). Bizzell OM (1966) Early history of radioisotopes from reactor. Isot. Rad.Tech., 4, 25-32. Bohr N (1936) Neutron capture and nuclear constitution, Nature 137, 344-348. Botros N, El-Garhy M, Abdulla S and Aly HF (1986) Comparative studies on the development of W-188/Re188 generator. Isotopenpraxis, 22, 368–371. Boyd RE (1982) Molybdenum-99: Technetium-99m Generator. Radiochimica. Acta, 30,123-128. Brown LC (1971) Chemical processing of cyclotron produced 67Ga. Int. J. Appl. Rad. Isot., 22,710–713. Brown LC (1972) Cyclotron processing of carrier-free 111In. Int. J. Appl. Rad. Isot. 23, 57-63. Brown LC and Callahan AP (1972) The separation and purification of carrier-free copper isotopes for medical use. Int. J. Appl. Radiat. Isot., 23, 535-539. Callahan AP, Rice DE and Knapp FF Jr. (1989) NucCompact, 20, 3. Cohen BL (1977) High level radioactive waste from light water reactors, Reviews of Modern Physics,49, 1–20. Cole WC, DeNardo SJ, Meares CF, McCall MJ, DeNardo GL, Epstein AL, O'Brien HA and Moi MK (1986) Serum stability of 67Cu chelates: comparison with 111in and 57Co. Int. J. Radiat. Appl. Instrum. Part B, Nucl. Med. Biol. 13, 363. Dadachova E, Mirzadeh S, Lambrecht RM, Hetherington EL and Knapp FF, Jr. (1994) Separation of Carrier-free Holmium-166 from Neutron-irradiated Dysprosium Targets. J. Analyt. Chem., 66, 4272. Dadachova E, Mirzadeh S, Lambrecht RM, Hetherington E and Knapp FF, Jr. (1995a) Separation of carrier-free 166 Ho from DyaO^ targets by partition chromatography and electrophoresis, J. Radioanalylt. Nucl. Chem. -Letters, 199, 115-123. Dadachova E, Mirzadeh S. Lambrecht RM (1995b) Tungstate-ion-alumina interaction in a 188W —>188Re biomedical generator, J . Phys. Chem., 99, 10976–10981. Dadachova E, Mirzadeh S, Smith SV, Knapp FF, Jr., Hetherington EL (1997) Radiolabeling antibodies with Holmium-166, Appl. Rad. Isot. 48, 477–481. Dasgupta AK, Mausner LF and Srivastava SC (1991) A new separation procedure for 67Cu from proton irradiation of Zn, Int. J. Radiat. Appl. Instrum. Part A, Appl. Radiat. Isot. 42, 371–376. DeNardo SJ, DeNardo GL, Kukis DL, Shen S, Kroger LA, DeNardo DA, Goldstein DS, Mirick GR, Salako Q, Mausner LF, Srivastava SC and Meares CF (1999) 67Cu-2TT-BAT-Lym-l pharmacokinetics, radiation dosimetry, toxicity and tumor regression in patients with lymphoma, J. Nucl. Med. 40, 302–310.

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DeNardo G, DeNardo S, Kukuis D, Diril H, Suny C and Meares C (1991) Strategies for enhancement of radioimmunotherapy. Int. J. Radial. Appl. Instrum. Part B, Nucl. Med. Biol. 18, 633-640. Deshpande S, DeNardo SJ, Meares CF, McCall MJ, Adams GP, Moi MK and DeNardo GL (1988) Copper-67 labeled monoclonal antibody Lym-1, a potential radiopharmaceutical for cancer therapy: labeling and biodistribution in RAJI tumored mice. J. Nucl. Med. 29, 217-225. Dickson DPE and Berry FJ, (eds) (1986) Mossbauer Spectroscopy, Cambridge University Press, Cambridge, U.K., pp 21-69. Ehrhardt GJ, Ketering AR, Turpin TA, Razavi MS, Vanderheyden JL and Fritzberg AR (1987) An improved tungsten-188/rhenium-188 generator for radiotherapeutic applications. J. Nucl. Med., 28, 656. Ehrhardt GJ, Turpin TA, Razavi MS, Vanderheyden JL, Su FM and Fritzberg AR (1990) in: Technetium and Rhenium in Chemistry and Nuclear Medicine 3, Nicolini M, Bandoli G and Mazzi U (ed's). Raven Press, New York, 631. Ehrhardt GJ, Ketering AR and Liang Q (1992) Improved 188W/188Re zirconium tungstate gel radioisotope generator chemistry, Radioactivity and Radiochemistry, 3, 38-41. Evans CC and Stevenson J( 1956) Improvements in or relating to production of radioactive Iodine -131. British Patent 763 865. Evans CC and Stevenson J (1957) Production of radioactive phosphorous, British Patent 765,489. Firestone RB, Shirley VS, Baglin CM, Chu F and Zipkin J (eds )(1996) Table of Isotopes, 8th edition, John Wiley and Sons, New York. Fritze K (1964) The preparation of high specific activity Copper-64. RadioChim. Acta, 3, 166-167. Glasstone S and Sesonske A (1963) Nuclear Reactor Engineering, Van Nostrand, Princeton. Grant PM, Miller DA, Gilmore JS and O'Brien HA Jr.(l 982) Medium energy spallation cross-sections. !. RbBr Irradiation with 800-MeV Protons. Int. J. Appl. Radiat. Isot., 33, 415-417. Green MA (1986) A potential copper radiopharmaceutical for imaging the heart and brain: copper-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone). Int. J. Radiat. Appl. Instrum. PartB. Nucl. Med. Biol. 14,59-61. Green MA, Klippenstein DL and Tennison JR (1988) Copper(II) Bis (thiosemicarbazone) complexes as potential tracer for evaluation of cerebral and myocardial blood flow with PET. J. Nucl. Med.. 29, 1549–1557. Griffiths GL, Goldenberg DM, Sharky RM, Knapp FF, Jr., Callahan AP, Tejada G and Hansen HJ (1984) Radionuclide Generators, Knapp and Butler (ed's), ACS Advances in Chemistry, Series No. 214, American Chemical Society, Washington, D.C, pp 33–37. Handley TH (1963) Di-n-butyl phosphorothioic acid as an extractant for metal ions. Anal. Chem. 35, 991-995. Hasany SM (1980) Extraction separation and preconcentration of divalent copper from aqueous-media. Radiochim. Acta, 27, 43-45. Hodgon PE (1967) The optical model of nucleon-nucleus interaction, In Annual Review of Nuclear Science 17, ppl-32 Hoeschele JD, Butler T, Roberts J, and Guyer C (1982) Analysis and refinement of the microspace synthesis of the !95mPt-labeled antitumor drug, cis-DDP, Radiochimica Acta, 31, 27-36. Hupf HB (1976) Production and purification of radionuclides, In Radiopharmacy, Tubis M and Wolf A (eds) John Wiley & Sons, New York, 225-253. Kadina G, Tulskaya T, Gureev E, Brodskaya G, Gapurova O and Drosdovsky B (1990) Technetium and

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Rhenium in Chemistry and Nuclear MedicineB. Nicolini M, Bandoli G and Mazzi U (eds), Raven Press, New York, 6353. Kamioki H, Mirzadeh S, Lambrecht RM, and Knapp FF Jr. and Dadachova E (1994) !88W-^188Re Generator for biomedical applications. Radiochimica Acta, 65, 39-46. Knapp FF, Jr., CalJahan AP, Beets AL, Mirzadeh S and Hsieh B-T (1994) Processing of reactor-produced Tungsten-188 for fabrication of clinical scale alumina-based Tungsten- 188/Rhenium-188 generators. Appl. Rad. and hot., 45, 1123-1128. Knapp FF, Jr., Beets, AL, Guhlke S, Zamora PO, Bender H, Palmedo H and Biersack H-J (1997) Development of the alumina-based Tungsten-188/Rhenium-188 generator and use of Rhenium-188-labeled radiopharmaceuticals for cancer Treatment. Anticancer Research, 17, 1783. Lagunas-Solar MC and Jungerman J A (1979) Cyclotron production of carrier free 55Co, a new positron-emitting label for bleomycin. Int. J. Applied Radiation and Isotopes, 30, 25-32. Lewis RE, and Eldridge JS (1966) Production of 70-day tungsten-188 and development of a 17 hour rheniurn188 radioisotope generator. J. Nud. Med., 7, 804. Liverhaut SE (1960) Elementary Introduction to Reactor Physics, Wiley Interscience, New York Mani RS and Majali AB (1966) Production of carrier free i2P. Indian Journal of Chemistry, 4, 391, Martell AE and Smith RM (1974) Critical Stability Constants, Vol. 1, Arnino Acids, Plenum Press, New York. Mausner LF, Mirzadeh S and Ward TE (1985) Nuclear Data for Production of 117mSn for Biomedical Application, In Proceedings of the International Conference on Nuclear Data for Basic and Applied Science, Santa Fe, New Mexico, May, 733-737. Mausner LF, Mirzadeh S and Srivastava SC (1992) Improved specific activity of reactor produced 117mSn with the Szilard-Chalmers process. Int. J. Appl. Radiat. hot. 43, 1117-1122. Mausner LF (1999) Radiochemical laboratory . In McGraw Hill Encyclopedia of Science & Technology, 9th edition, McGraw Hill New York. Maziere B, Stulzaft O, Verret JM, Comar D and Syrota A (1983) 55Co- and 64Cu-DTPA: New radiopharmaceuticals for quantitative tomocisternography. Int. J. Appl. Radiat. hot. 34, 595–601. Mikheev V, Popvich VB, Rumer IA, Savelev GI and Volkova NC (1972) Re-188 generator Isotopenpraxis, 8, 248-250. Mirzadeh S, Parekh PP, Katcoff S and Chu YY (1983) Cross-section systematics for nuclide production at a medium energy spallation neutron facility, Nuclear Instruments and Methods, 216,149-154, Mirzadeh S, Mausner LF and Srivastava SC (1986) Production of no-carrier-added 67Cu. Int. J. Radiat, Appl. lustrum. Part A, Appl. Radiat. hot. 37, 29-36. Mirzadeh S and Knapp FF Jr. (1992) Spontaneous electrochemical separation of Carrier-free copper-64 and copper-67 from zinc targets. Radiochimica Acta, 57, 193-199. Mirzadeh S, Schenter RE, Callahan AP and Knapp Jr FF (1992) Production Capabilities in U.S. NuclearReactors for Medical Radioisotopes. ORNL/TM-12010, National Technical Information Services, Springfield VA. Mirzadeh S and Packard AB (1995) Synthesis of AZT-Pt(terpy) - A potential compound for radiotherapy of AIDS. Proceedings of Symposium on Advances in Bioconjugate Chemistry. 210th ACS National Meeting, Chicago, 111, Aug. 20-24.

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Mirzadeh S and Knapp FF, Jr. (1996) Biomedical radioisotope generator systems. J. Radioanalyrical Nucl. Chem, 203, 471-488. Mirzadeh S, Knapp FF, Jr. and Lambrecht RM (1997a) Burn-up cross-section of W-l 88, Radiochimica Acta, 77, 99-102. Mirzadeh S, Knapp FF Jr., Alexander CW and Mausner LF (1997b) Evaluation of neutron inelastic scattering for radioisotope production. Appl. Radial. I sot., 48, 441-446. Mirzadeh S (1998, unpublished). Mirzadeh S and Walsh P (1998) Numerical evaluation of the production of radionuclides in a nuclear reactor. Part I&II. Applied Radiation and Isotopes, 49, 370- 383. Mirzadeh S, Du M, Beets AL and Knapp FF, Jr. (2000) Thermoseperation of neutron irradiated tungsten from Re and Os. Industrial and Engineering Chemistry Research. 39, 3169-3172. Mughabghab SF, Divadeenam M and Holden NE (1981) Neutron Cross-sections, Vol. 1, Part A; 2=1-60, Academic Press, New York. Mughabghab SF, Divadeenam M and Holden NE (1984) Neutron Cross-sections, Academic Press, New York. Mughabghab SF (1984) Neutron Cross-sections, Vol. 1, Part B; Z=6J-JOO, Academic Press, New York. Neirinckx RD (1977) Simultaneous production of Cu-76, Cu-64and Ga-67 and labeling of bleomycin with Cu67 and Cu-64. Int. J. Appl. Radiat. I sot., 28, 802-804. O'Brien HA Jr. (1969) The preparation of 67Cu from 67Zn in a nuclear reactor. Int. J. Appl. Radiat. Isot., 20, 121 124. Pastakia B, Lieberman LM, Gatley SJ, Young D, Petering DH and Minkel D (1980) Tissue distribution of copper-labeled 3-ethoxy-2-oxobutyraldehyde bis (thiosemicarbazone)(Cu-64 KTS) in mice and rats. J. Nuclear Medicine. 21, 67-70. Polak P, Geradts J, Van der Vlist R and Lindner L (1986) Photonuclear production of 67Cu from ZnO targets. Radiochimica Acta 40, 169-174. Richards P (1966a) Nuclide generators. In Radioactive Pharmaceuticals, Symposium #6, Conf. 65111 L US Atomic Energy Commission, Washington, DC. Richards P (1966b) The Technetium-99m Generator, in Radioactive Radiopharmaceutica\s, PROC. CONF.651111, National Bureau of Standards, Springfield, Virginia, pp 323-334 Richards P (1966c) Nuclide Generators, in Radioactive Radiopharmaceuticals, PROC. CONF.-651111, National Bureau of Standards, Springfield, Virginia, pp 155-163. Roberts JC, Figard SD, Mercer-Smith JA., Svitra ZV, Anderson WL and Lavallee DK (1987) Preparation and characterization of copper-67 porphyrin-antibody conjugates. J. Immunological Meth. 105, 153-159. Roberts JC, Newmyer SL, Mercer-Smith JA, Schreyer SA and Lavallee DK (1989) Labeling antibody with copper radionuclides using N-4-Nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine. Int. J. Radiat. Appl. Instrum. Part A, Appl. Radiat. I sot. 40, 775-781. Smith SV, Di Bartolo N, Mirzadeh S, Lambrecht RM and Knapp FF Jr. (1995) [166Dy] Dysprosium/[166Ho] Holmium In Vivo generator, Applied Radiation and Isotopes 46, 759-764. Srivastava SC, Atkins HL, Krishnamurthy GT, Zanzi I, Silberstein EB, Meinken G, Mausner LF, Swailem F, D'Alessandro T, Cabahug CJ, Lau Y, Park T and Madajewicz S (1998) Treatment of metastatic bone pain with tin-117m stannic diethylenetriaminepentaacetic acid: a phase I/II clinical study. Clinical Cancer Research 4, 61–68.

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Subramanian G (1976) Radioisotope Generators. In Radiopharmacy, Tubis M and Wolf W (eds), John Wiley & Sons, New York, 255-277. Szilard L and Chalmers TA (1934) Chemical separation of the radioactive element from its bombarded isotope in the Fermi effect. Nature, 134, 462. Tucker WD, Greene MW, Weiss AJ and Murrenhoff AP (1958) Methods of preparation of some carrier-free radioisotopes involving sorption on alumina, Transactions of the American Nuclear Society 1,160-161. Weinreich R, editor (1992) Targetry '91. Proceeding of the IVth International Workshop in Targetry and Target Chemistry. Paul Scherrer Institute 92-01, Villigen Switzerland. Willins JD and Sgouros G (1995) Modeling analysis of Pt-195m for targeting individual blood-born cells in adjuvant radioimmunotherapy. J. Nuclear Medicine. 36, 315-319. Winsche WE, Stang LG and Tucker R (1951) Production of 132I. Nucleonics 8, 14.

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4. CHEMISTRY OF NITROGEN-13 AND OXYGEN-15 JOHN C. CLARK AND FRANKLIN I. AIGBIRHIO Wolfson Brain Imaging Centre, University of Cambridge, Box 65, Addenbrooke's Hospital, Cambridge, CB2 2QQ, United Kingdom

INTRODUCTION The positron-emitting radioisotopes, nitrogen-13 (half-life; 9.965 min, 100% p+, decay) and oxygen-15 (half-life; 2.07 min, 99.9% $+, decay) present many unique challenges with regard to their production, chemical incorporation into biologically useful tracers and their application in biomedicine. Due to their short half-lives, very rapid techniques for their synthesis and ingenious methods for their application are required. Their use, however, can offer significant advantages. They provide access to studying a wide range of important biological processes that involve compounds containing nitrogen or oxygen (Table 1). Biologically important molecules that can be labelled include oxygen, water, ammonia and amino acids. Their very short half-life also has the advantage that repeated studies may be performed on the same individual within a short period of time. In this chapter we will outline the key radiochemistry and methods required for the application of nitrogen-13 and oxygen-15 to biochemical and biomedical studies, including radionuclide production methods and the radiochemical synthesis of their precursors and radiotracers. Table 1. Key nitrogen-13 and oxygen-15 radiopharmaceuticals and their applications Radiopharmaceuticals

Application

Reference

[ N] Ammonia

Cerebral and myocardial blood flow

Phelps et al., 1977', Schelbert, 1986

Pulmonary perfusion/ventilation and nitrogen fixation studies

Schuster, 1998

[150]02

Tissue oxygen metabolism

Ter-Pogossian & Herscovitch, 1985

[I5O]H2O

Cerebral and myocardial blood flow

Ter-Pogossian & Herscovitch, 1985

Blood volume studies

Ter-Pogossian & Herscovitch, 1985

Cerebral blood flow

Herscovitch et al., 1987

5

O]Butanol

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

HANDBOOK OF RADIOPHARMACEUTICALS

120

THE CHEMISTRY OF NITROGEN-13 Nitrogen-13 has the distinction of being one of the earliest positron-emitting radionuclides to be produced. It was first prepared by the irradiation of boron nitride with a-particles using the 10B(a, n)13N reaction (Joliot & Curie, 1934). This was then used to prepare the first and still the most important nitrogen-13 radiotracer, [13N]ammonia, by heating the boron nitride with sodium hydroxide. Using cyclotron production techniques very high radioactivities (> 50GBq) and high specific activities (> 400GBq/umol) can now be produced. This has enabled a significant range of nitrogen-13 labelled organic and inorganic molecules to be prepared, most significantly amino acids using biosynthetic techniques. PRODUCTION OF NITROGEN-13 Nitrogen-13 can be produced from targets containing gases, liquids or solids using compounds of boron, carbon, nitrogen or oxygen with the appropriate accelerated particle (Table 2). The range of nuclear reactions that have been used to produce nitrogen-13 include 12C(p,7)13N, 13C(p,n)13N, l4N(p,pn)13N, 10 B(a,n)13N and 14N(n,2n)13N. However, the two most useful are the 12C(d,n)13N and 16O(p,a)13N reactions (Whitehead & Foster, 1958; Straatman, 1977; Ferrieri & Wolf, 1983). Table 2. Methods for the production of nitrogen-13. Target Material

Nuclear Reaction

In-Target product

Post irradiation treatment

Final product

CO2 (trace N2) Graphite (CO2 sweep with trace N2) Charcoal (He sweep)

12

C(d,n)13N

[13N]N2

CO2 absorption

[ 13 N]N 2

C(d,n)13N

Trapped [13N]CN

On-line release as [ 13 N]N 2 through graphite oxidation to CO

[ I 3 N]N 2

12

C(d,n)13N

[13N]N2 and trapped [13N]CN

On-line release

[13N]N2

13

C(p,n)13N

Trapped [13N]CN

On-line release in He sweep as [13N]N2 using high intensity beam (>10nA)

[ 13 N]N 2

13

C enriched charcoal

12

With microwave excitation in H2 plasma

3

N]NH3

16

O(p,a)13N

[ IJ N]NH 3

Radiochemical purification

[ U N]NH 3

H20

16

O(p,a)13N

[13N]NH/ + [13N]NO3- + [13N]N02-

DeVarda's alloy and NaOH

[I3N]NH3

Aq NaNO3

14

N(n,2n)13N

[13N]NH3

A14C3

I2

C(d,n)13N

Matrix trapped [13N]

Acid reduction and radiochemical separation of [13N]CH3NH2

[13N]NH3

CH4 (flowing)

12

[13N]NH3 + [13N]CH3NH2 + [13N]HCN

radiochemical purification

[ I3 N]NH

H2O/ethanol, H 2 or CH4 overpressure

C(d,n)13N

[13N]NH3

CHEMISTRY OF NITROGEN-13 AND OXYGEN-15

121

[13N]NITROGEN, [13N]N2 Early methods used for the preparation of [13N]nitrogen were based on deuteron irradiation of targets containing carbon-12 sources. Continuous productions were obtained by using carbon dioxide as the target material as well as the sweep gas for the removal of the [13N]nitrogen (Ruben et al., 1940; Clark & Buckingham, 1975). Higher yielding batch-wise productions were obtained using graphite as the target material (Ruben et al., 1940; Clark & Buckingham, 1975). Both methods produced [13N]nitrogen in high radiochemical purity. Later methods involved proton irradiation of targets containing carbon enriched with carbon-13 (Wolk et al., 1976; Ferrieri et al., 1983) aqueous ammonium hydroxide (Suzuki et al., 1977) and ammonia (Parks & Krohn, 1978). Radiosynthesis from [13N]ammonia can also be used through reaction with sodium hypobromite (Scheme 1) (Lindner et al., 1979; Vaalburg et al., 1981).

2 13 NH 3

+ 3 NaOBr

-

[13N]N2

+

3NaBr

+

3H2O

Scheme 1. Synthesis of [13N]nitrogen from [13N]ammonia. The 14N(p,pn)13 N nuclear reaction that occurs concurrently in a nitrogen gas target during the production of [l!C]carbon dioxide using l4N(p,a)11C process has also been used (Le Bars, 2001). [13N]Nitrogen was obtained at high purity through passage of the target gas through a series of traps; initially an AscariteR trap to remove the [11C]carbon dioxide and short lived oxygen-14 products, followed by a copper oxide furnace to convert [HC]carbon monoxide to [11C]carbon dioxide and finally another AscariteR trap. Due to its low reactivity there have been no significant radiosynthetic applications of [13N]nitrogen. However, its inert property makes it an ideal tracer for determining regional pulmonary ventilation in man by PET (Schuster, 1998). This represents the principal imaging application of [13N]nitrogen. However, there are a range of studies in which [13N]nitrogen has been used to investigate plant and micro-organism physiology, especially nitrogen fixation processes (Ruben et al., 1940; Nicholas et al., 1961; Thomas et al., 1977; Meeks et al., 1978). [13N]NITRATE, [13N]NO3 AND [13N]NITRITE, [13N]NO2 [13N]Nitrate can be readily produced at high yields by the proton irradiation of oxygen-16 water (Krizek et al., 1973; Welch & Straatman, 1973). In general, the [l3N]nitrate component increases with relation to the other target products, [13N]ammonia and [l3N]nitrite, with increasing irradiation (Parks & Krohn, 1978; Root, 1981). The [13N]nitrate can amount to as high as 85% of the radiochemical products. The product is commonly obtained by passing the target water through a cation exchange column to remove [13N]ammonium and vanadium-48 contamination from the titanium beam entry window (Wieneke & Nebeling, 1990). Sulphuric acid is then added to the target water to remove nitrous acids. An alternative method for purification is based on preparative HPLC using aqueous perchloric acid as the eluent (Wieneke & Nebeling, 1990). [13N]Nitrite, which is also produced through proton irradiation of oxygen-16 water target, can be obtained in high purity through HPLC separation of the target product (Wieneke & Nebeling, 1990). However higher yields can be obtained by reduction of the co-produced [13N]nitrate to [°N]nitrite by

122

HANDBOOK OF RADIOPHARMACEUTICALS

passage through a copperised cadmium reduction column (McElfresh el al., 1979; Chasko & Thayer, 1981; Vavrek & Mulholland, 1995). Generation of [ 13 N]nitrite in situ from [13N]ammonia using copper dust has also been used (Pettit et al., 1977). [13N]NITROUS OXIDE [13N]N2O The chemical synthesis of [13N]N2O has been reported (Nickles et al., 1978). The synthesis was based on the classical decomposition of NH4NO3but with some significant modifications. [13N]NO3 produced by the proton irradiation of water was introduced into a solution of (NH4)2SO4 in concentrated H2SO4. On heating the mixture [13N]N2O was released in good yield and purity. [13N]AMMONIA, [13N]NH3 [13N] Ammonia is by far the most important [13N]radiolabelled compound used in PET imaging studies. It is a key reagent for the introduction of nitrogen-13 into more complex structures. It is a highly diffusible tracer, which after intravenous injection, is efficiently extracted into tissue, where it remains for a considerable time due to various fast metabolic processes. Based on these properties it has become an important tracer for regional blood flow in tissues (Phelps et al., 1977; Schelbert, 1986) especially for quantitative determinations of myocardial perfusion (Wiyns & Camici. 1997). [ I3 N]Ammonia was first produced by heating ex-particle irradiated boron nitride in sodium hydroxide (Joliot & Curie, 1934). However, production in significant amounts was later achieved by the irradiation of metal carbides with deuterons, dissolving the target in boiling HC1 and then distilling the [13N]ammonia produced by the addition of NaOH (Welch & Lifton, 1971; Hunter & Monahan, 1971). Another method was based on the irradiation of methane gas with deuterons to form [ 13 N]ammonia directly in the target (Tilbury et al., 1971; Straatman & Welch, 1973). However, the method gives low yields due to the production of significant amounts of alkyl[ 13 N]nitrogen compounds and other polymeric products of radiolysis. The method of choice and most widely used is the proton irradiation of natural water target (Krizek et al., 1973; Welch & Straatman, 1973; Tilbury & Dahl, 1979). [ 13 N]Ammonia is formed as the primary product by the abstraction of hydrogen from the water (Scheme 2). However, as the radiation dose to the target is increased radiolytic oxidation occurs producing oxo anions of nitrogen, mainly nitrates and nitrites. 13

N

13

NH3

+

H-OH +

HOH 13

NH4+

"~

13

*•

13

*•

NH3

13

Hydrogen abstration

NH4+

NO3

OH-

+

13

NO2~

Hydration

Radiolytic oxidation

Scheme 2. Chemical reactions occurring during proton irradiation of an oxygen-16 water target. These oxo anions can be removed from the irradiated water with an ion exchange resin to leave [13N]ammonia. However, to increase the radiochemical yield of [13N]ammonia. a more effective method involves the chemical reduction of these oxo anions to [ 13 N]ammonia. Reducing reagents used for this

CHEMISTRY OF NITROGEN-13 AND OXYGEN-15

122

process include DeVarda's alloy in aqueous sodium hydroxide (Vaalburg et al., 1975) and titanium (III) chloride or titanium (III) hydroxide (Ido & Iwata, 1981). The [13N]ammonia can then be removed from the alkaline reducing mixture with a stream of helium gas and collected in slightly acidic sterile, apyrogenic water or saline ready for use. The formation of the oxo anions in the target can be prevented by the addition of free radical scavengers to the target water resulting in only [13N]ammonia being produced. These scavengers are commonly ethanol, acetic acid (Weiland et al., 1991) or hydrogen (Mulholland et al., 1989; Berridge & Landmeir, 1993). The use of methane over pressure in natural water target irradiated with protons has also been found to be very effective in producing high purity [13N] ammonia (> 99 %) even at very high beam currents (Krasikova et al., 1999). It has been shown that the use of a cryogenic target containing frozen water for the

16

O(p,a)13N reaction significantly reduces

the radiolytic formation of oxo anions (Firouzbakht et al., 1999). This results in the formation of [13N]ammonia at more than 95 % purity. A significant advantage of in-target methods for [13N]ammonia production is it enables production of higher specific activities since there is no carrier contamination from the use of reducing reagents (Suzuki et al., 1999). NITROGEN-13 LABELED AMINO ACIDS An extensive range of nitrogen-13 labelled amino acids have been prepared (see Table 3). These compounds are of particular interest for imaging and for the determination of protein synthesis rates, particularly in tumours (Cooper & Gelbard, 1981; Cooper et al, 1985; Fowler & Wolf, 1986). Biosynthetic methods using enzymes that are responsible for ammonia metabolism in living systems are mainly used for their synthesis. These enzymes enable rapid regio and stereospecifc syntheses to be performed under mild aqueous reaction conditions. They are often utilised after being immobilised on a solid support, commonly cyanogen bromide activated

sepharose

or porous

derivatized

(N-

hydroxysuccinimide) silica beads. This has the advantage of preventing antigenic and pyrogenic macromolecules contaminating the final products and enabling the enzyme to be reused for several syntheses. The most versatile enzyme system used is glutamate dehydrogenase (GAD) which catalyses the reductive amination of alpha-keto acids using [13N]ammonia to produce L-[13N]amino acids (Scheme 3) (Cooper & Gelbard, 1981).

COO

COO

CH2 I CH2 + 13NH3 + NAD(P)H + 2H+

CH2 I CH2

1

I c=o

I

.

+ H2O + NAD(P)

Glutamate

COO a-Ketoglutarate

COO" L- [13N]-Glutamate

NAD(P) = nicotinamide adenine dinucleotide phosphate Scheme 3. Synthesis of L-[13N]-Glutamate using glutamate dehydrogenase

124

HANDBOOK OF RADIOPHARMACEUTICALS

Other key enzymes include glutamine synthetase (GS), glutamate-oxaloacetate transaminase (GOT), asparagine synthetase (AS), glutamate-pyruvate transaminase (GTP) and phenylalanine dehydrogenase (PAD).

Table 3. Nitrogen-13 labelled amino acids produced using biosynthetic methods 13

Precursor

13

Enzyme

Reference

L-[ NJAlanine

Pyruvate

[13N]NH3

GAD

Cohen et al.. 1974; Coope &Gelbard, 1981;

N Labelled Amino Acid

N Reagent

Baumgartner et al., 1981 Pyruvate

L-[13N]Glutamate

GTP

Cohen et al., 1974

L-[13N]a-Aminobutyric acid

a-ketobutyrate

[13N]NH3

GAD

Cooper & Gelbard, 1981

L-amide-[13N] Asparagine

Aspartate

[13N]NH3

AS

Gelbard et al., 1974; Majumder et al., 1977.

L-[I3N]Aspartic acid 13

L-amide-[ N]Glutamine

Oxalacetate L-Glutamate

L-[13N]Glutamate 13

[ N]NH3

GOT

Cooper & Gelbard, 1981

GS

Cooper & Gelbard, 1981; Straatmann & Welch, 1973; Gelbard et al., 1975

L-[l3N]Glutamic acid

a-Ketoglutarate

[13N]NH3

GAD

Gelbard et al., 1974: Suzuki et al., 1982

L- [13N] (3R,S)-Isoleucine

D,L-cc-keto-pmethyl-n-valerate

[13N]NH3

GAD

Barrio et al., 1982

L-[13N]Leucine

a-ketoisocaprate

[I3N]NH3

GAD

Gelbard et al., 1974; Straatmann & Welch, 197

L-[l3N]Methionine

a-keto-y-methiolButyrate

[13N]NH3

GAD

Gelbard et al., 1974

L-[13N]Phenylalanine

phenylpyruvate

[ I3 N]NH 3

PAD

Gelbard et al., 1990

L-[13N]Tyrosine

L-[13N]Valine

13

phenylpyruvate

L-[ N]Glutamate

GOT

Gelbard & Cooper. 1979

p-hydroxyphenylpyruvate

L-[l3N]Glutamate

AS

Gelbard & Cooper, 1979;

p-hydroxyphenylpyruvate

[13N]NH3

PAD

Gelbard et al., 1990

a-Ketoisovalerate

[13N]NH3

GAD

Gelbard et al., 1974

Gelbard et al., 1990

CHEMISTRY OF NITROGEN-13 AND OXYGEN-15

125

Some amino acids have also been prepared using synthetic organic methods. [13N]Asparagine has been prepared by reacting L-a-N-t-Boc-t-Bu-aspartate

(activated

with AMiydroxysuccinimide) with

[13N]ammonia obtaining radiochemical yields of 30-40 % (Elmaleh et al., 1979) whereas only yields of 10-20 % are obtained using enzymes. [13N]Gamma-aminobutyric acid has also been produced from [13N]ammonia in up to 65 % yield by the amination of the organoborate triisopropylsilyl 4(dimethylboro)butanote (Kabalka et al., 1992). NITROGEN-13 LABELLED AMINES Due to the importance of amines in various physiological processes including function as neurotransmitters and neuromodulators, they have been of interest for labelling with nitrogen-13. An early series of studies involved biological studies in rats using [13N]amines of the form RCH213NH2 (Lade et al., 1979). Later [13N]amphetamine was produced via [13N]imines synthesised from [13N]ammonia (Finn et al., 1981). However, this method gave low radiochemical yield and specific radioactivity (3.5 % and 8 Ci/mol respectively). The potent nucleophilic reagent [13N]hydroxylamine was synthesised from [13N]nitrite and sodium bisulphite in good radiochemical yields of 50-90 % (Kaseman et al., 1984). Using a Hoffman rearrangement of the amide [13N]phenylpropionamide the neuromodulator [13N]-(3-phenethylamine has been synthesised. [13N]Phenylpropionamide was synthesised by reacting phenylpropionyl chloride and aqueous [13N]ammonia solution (Tominaga et al., 1985). However good yields of [!3NJ-p-phenethylamine, 50 %, could only be obtained under carrier-added conditions, with yields reduced to ca 5 % at high specific activities. This method was later modified by utilising lithium aluminium hydride for reductions (Scheme 4) (Tominaga et al., 1986).

Na2CO3 — +

n

C013NH2

LiAlH4 +

13

H2C13NH2

CH,

Scheme 4. Synthesis of [13N]-|3-phenethylamine This improved method gave high radiochemical yields (52 %) of [13N]-p-phenethylamine at high specific activities and also enabled [13N]octylamine to be synthesised in good yields (59 %). The application of organoboranes provides a very convenient method for the preparation of [!3N]amines. Amination of organoboranes with [13N]ammonia can be performed under mild conditions using in situ preparation of [13N]chloramines. This was then used to produce the amines l-[13N]aminodecane (Scheme 5), l-[!3N]aminooctane, l-[13N]aminohexane (Kothari et al., 1986) and [13N]putrescine (Kabalka et al., 1992). The mild conditions also enabled this method to be used for the preparation of functionally substituted amines e.g. [13N]dopamine (Kabalka et al., 1992).

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126

13,

3 C8H17-CH=CH2 + BH3

•

THF

1 0H2211)36 3

NaOCl

10H21

13

NH2

Scheme 5. Synthesis of l-[13N]aminodecane This method was also refined to simplify the purification of the [I3N]amine product by using precursors based on polymeric borane reagents (Kabalka et al., 1993) (Scheme 6).

13

NH,

NaOCl

OH

O

OH [ N]Gamma-aminobutyric acid

[r!3 NJDopamine

NH, 13 [r13 N]Putrescine

Scheme 6. Synthesis of nitrogen-13 labelled amines using organoborane polymers OTHER NITROGEN-13 RADIOTRACERS A method for the on-line production of [13N]nitric oxide has been developed (McCarthy et al., 19%). This is based on the selective chemical clean-up of the target output obtained from deuteron irradiation of graphite heated in a stream of pure oxygen (Dence et al., 1994). The target output product which consist mainly of [13N]nitric oxide and [13N]nitrogen dioxide is passed through a column of o-tolidine packed in silica gel (Kitagawa tube®) which selectively removes the [l3N]nitrogen dioxide. [13N]Nitric oxide has become of research interest for investigating the pharmacology of nitric oxide, which is gaining recognition as a therapeutic agent for various types of pulmonary hypertension (Lunn, 1995). [l3N]Urea has been prepared by boiling [13N]ammonia in the presence of ammonium cyanate (Krizek et al., 1977]. Unlabeled urea was then added to the reaction solution which was purified using a cation exchange column to obtain [13N]urea in a yield of 56 %. N-nitrosureas have been labelled with nitrogen-13 and are of interest due to their biological properties as carcinogens as well as chemotherapeutic agents. The anti-tumour drug CCNU (l-(2-chlorethyl)-3cyclohexyl-3-cyclohexy-l-nitrosourea) was labelled by reacting [13N]nitrous acid produced from [13N]ammonia with a urea precursor (Pettit et al., 1975). However, yields of the product l-(2chloroethyl)-3-cyclohexyl-l-[13N]nitrosourea were very low, 0.3-1.5 %. An improved method based on the in situ generation of [13N]nitrite from [13N]nitrate using copper dust was used to make l,3-bis(2-

CHEMISTRY OF NITROGEN-13 AND OXYGEN-15

127

chlorethyl)-l-[13N]nitrosourea (BCNU) with yields of 20-40 % (Pettit et al., 1977). This method was later optimised by the use of more concentrated carrier solutions to prepare the antibiotic [l3N]streptozotocin and [13N]nitrosocarbaryl in yields of about 20 % (Digenis et al., 1979) and the carbamate analog of BCNU, N-[13N]nitroso-N-chloroethyl-l-chloroethyl carbamate (BCNC) in yields of 10% (Digenis et al., 1981). A simple resin-supported method for the synthesis of [13N]nitrosamines and [13N]nitrosothiols from [13N]nitrites has been developed (Vavrek & Mulholland, 1995). [13N]Nitrite is trapped on an AGl(OH) anion exchange resin column (SAX) followed by either elution with a thiol solution to form a [13N]nitrosothiol in yields of 90%, or a secondary amine solution to form a [13N]nitrosamine in yields of greater than 30 %. The important anti-cancer drug, cisplatin (m-dichlorodiamineplatinum (II), cis-DPP) has been labelled by reacting a tetraiodo platinum salt with carrier-added [13N]ammonia (De Spiegeleer et al., 1986) (Scheme 7). The product was obtained in radiochemical yields of 27 % after HPLC purification using an anion exchange column.

AgNO3 9. K2PtI4 —-ZH^ ( i3 NH)ptI —^—^ (13NH3)2Pt(H2O)22+

NaCl

n

(13NH3)2PtCl2

Scheme 7: Synthesis of Cwplatin Significantly higher radiochemical yields of 80 % were obtained using a modified method based on performing the reaction with the tetraiodo platinum salt attached to a strong anion exchange resin (Holschbache et al., 1996). The tetrapeptide H-Tyr-D-Met(O)-Phe-Gly-NH2 has been labelled in the amide position by heating its activated ester with [13N]ammonia (Kiso et al., 1991). Using biosynthetic methods based on the animation with [13N]ammonia with the enzyme transglutaminase a range of polypeptides including vasoactive intestinal peptide, neuropeptide Y, secretin, glucagons and insulin have also been labelled, however usually in rather low yields ca 3 % (Landais et al., 1993). QUALITY ASSURANCE METHODS FOR [13N]AMMONIA Due to the importance of [13N]ammonia as a diagnostic tracer for in vivo PET imaging (Wiyns & Camici, 1997) there are guidelines for quality control in both the European and US Pharmacopoeia. The main radionuclide contamination from the production of [13N]ammonia from the proton irradiation of water especially with increasing dose is fluorine-18 (t1/2 = 109.8 min) from the l8O(p,n)18F nuclear reaction. Fluorine-18 and cationic radionuclidic impurities are eliminated if the [13N]ammonia is prepared via reduction of the 13NO2 and 13NO2 and distillation. For in-target production methods these contaminates can be removed through the trapping of [13N]ammonia on a cation exchange column, followed by fractional desorption.

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HANDBOOK OF RADIOPHARMACEUTICALS

There is potential for chemical impurities when [13N]ammonia is produced using reduction methods. Traces of the reducing reagents may be carried over as spray during purification of the [l3N]ammonia by distillation. The presence of titanium from the target construction materials can be tested with spot tests (Feigl & Oesper, 1972) or colorimetric assays (Muller, 1967). Aluminium and copper from DeVarda's alloy can be detected using photometric methods, e.g., Spectroquant® from Merck. The radiochemical purity can be readily determined by radio-HPLC using cation exchange columns (Nieves et al., 1986) or by TLC (Galley & Shea, 1991).

THE CHEMISTRY OF OXYGEN-15 With its 2.07 min half-life it was not surprising that early investigators in tracer methodology (Siri, 1949; Kamen, 1957) dismissed the possibility that oxygen-15 would ever be used as a practical tracer in biology. In 1958 Ter-Pogossian and Powers (Washington University St Louis) were probably the first to demonstrate that oxygen-15 could be used to study regional tracer biology when they used [l5O]oxygen to assess its distribution in murine experimental neoplasms (Ter-Pogossian & Powers, 1958). Since that time many important advances have occurred both in preparation of a variety of radiotracers incorporating oxygen-15 and the ingenious design of the methodology for the biomedical exploitation of these radiotracers (Dyson et al., 1958; Dollery & West, 1960; Dyson et al., 1960; Ter-Pogossian et al., 1961; Lenzi et al., 1978; Segal et al., 1978; Ter-Pogossian & Herscovitch, 1985). PRODUCTION OF OXYGEN-15 Oxygen-15 (Half-life 2.07 min) can be produced using a variety of nuclear reactions (Beaver et al., 1976; Vera Ruiz & Wolf, 1977; Sajjad et al., 1984; Ruth, 1985; Sajjad et al., 1985; Krohn et al., 1986; Mullholland et al., 1990). The 14N(d,n)15O, 16O(p,pn)15O and 15N(p,n)15O are the most commonly used reactions. The latter became important with the advent of the commercially available low energy proton only cyclotrons (Wieland et al., 1986) whereas the 14N(d,n)15O has been exploited by the providers of low energy (~3-4 MeV) deuteron accelerators as so-called 15O-generators. A variety of accelerator target systems have been proposed and the chemical forms of [15O] generated in these systems have been explored by many (Table 4). Rarely, if ever, has the full [15O] radiolabelled product spectrum been analysed in any of the reports largely due to the significant difficulties in recovering all the radiolabelled products from the target. This is especially the case when nitrogen is irradiated with no additives. However, in practical terms when the additives described below are added to the nitrogen being irradiated, the product spectrum can usually be more readily analysed. IRRADIATION OF NITROGEN/OXYGEN MIXTURES The early reports on the production of [I5O]O2 described the use of air as the target material (Dyson et al., 1958; Welch & Ter-Pogossian, 1968). It was soon realised that this approach led to many undesirable chemical and radiochemical impurities, especially when the deuteron energy was not carefully controlled to minimise the production of other radionuclides generated by nuclear reactions at higher energies.

129

CHEMISTRY OF NITROGEN-13 AND OXYGEN-15 Table 4. Methods for the production of the oxygen-15 Target Material

Nuclear reaction

In-Target product

Post irradiation treatment

14

[15O]O2 + [15O]N2O [15O]NO2 + [15O]O3

Thermally cycled molecular sieve column

[15O]O2

Remove traces of

[15O]O2*

Remove traces of [15O]O2

[15O]CO2* 13 Trace [ N]N2

N(d,n)15O

N2(0.1-4%O2)

N2

4

N(d,n)150

14

N(d,n)150

5

O]CO2

(0.1-2%CO2)

Final product 5

O]N 2 O

remains from 12 C(d,n)l3N N(d,n)15O

[!5O]H2O

Remove traces of radiolytic NH3

[15O]H2O*

N(d,n)15O

[15O]CH3OH [15O]C2H5OH [15O]H2O + [15O]CH2O (trace)

Radiochemical separation

[15O]alcohols

[150]O2+O3

React with hot carbon

[I5O]CO2*

[15O]H2O

Remove [13N]

N2 (5% Ha)

14

N2 (5% CH4)

4

O,

16

0(p,pn)15O

H2O

16

O(p,pn)15O

Oxy-anions with ion exchanger 16 SiO2 (Ne/5%O2) O(p,pn)15O [15O]O2 + [15O]O3 *These [15O] tracers are most widely used

cryopurification

[15O]03

The use of N2/O2 mixtures in the range of 0.1 to 4 % oxygen concentration has been shown by many authors (Welch & Ter-Pogossian, 1968; Clark & Buckingham, 1975) to lead to far fewer impurities although there is a clear dependence on the product distribution on beam intensity. For example during the deuteron irradiation of a 4 % O2/ N2 mixture there being 3.3 and 0.7 % [15O]N2O and 96.7 and 99.3% [15O]O2 at 5 and 50 \iA respectively .

In the absence of oxygen the major [15O] labelled product of the irradiation of N2/CO2 mixtures in the range 0.25 to 5 % (Clark & Buckingham, 1975) is [I5O]C15O2. However, the radionuclidic impurity [13N]N2 is produced via the 12C(d,n)13N reaction and is difficult, if not impossible, to remove to obtain

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HANDBOOK OF RADIOPHARMACEUTICALS

high purity [15O]CO2. There is a clear dependence on the product distribution on beam intensity typically with a target containing 0.25 % CO2 yielding 2 and 0.5 % [15O]O2 and 98 and 99.5 % [I5O]CO2 at 5 fiA and 40 jiA respectivly. IRRADIATION OF NITROGEN/CARBON MONOXIDE MIXTURES The irradiation of essentially oxygen-free nitrogen in a recirculating target system containing an active carbon reaction tube at 900 °C yielded [15O]CO as the major product (Welch & Ter-Pogossian, 1968). Although the data as presented cannot be used to differentiate between the production of [15O]CO in the target and in the carbon reaction tube it seems likely that some [!5O]CO is produced directly in the target mainly as a result of radiolysis driven exchange reactions. IRRADIATION OF NITROGEN/HYDROGEN MIXTURES When nitrogen/hydrogen mixtures are irradiated the predominant oxygen-15 labelled product is [15O]H2O (Vera Ruiz & Wolf, 1978; Harper & Wickland, 1981). Further data on this system has enabled the optimisation of this method for the production of [15O]H2O whilst minimising the radiolytic production of NH3 (Hagami et al, 1986). IRRADIATION OF NITROGEN/WATER VAPOUR MIXTURES Observations on the irradiation of N2/H2O vapour mixture showed that a large fraction of the target output was of the form [15O]H2O (Clark unpublished observations). The results reported by (Welch et al.. 1969) when preparing [15O]H2O by isotopic exchange from [I5O]CO2 in a wet re-circulating system may have been confounded by the direct production of [15O]H2O in the target which would rapidly equilibrate with the water in the "bubbler" part of the circuit. IRRADIATION OF N2 TO PRODUCE [15O]N2O [15O]N2O has been observed as an in-target radiolysis product in the irradiation of N2/O2 mixtures (Clark & Buckingham, 1975; Diksic et al., 1983). By irradiating high purity nitrogen at a low -0.5 |iA beam current [15O]N2O can be cyclically recovered and purified using a zeolite column. The yield is reported to be 4 MBq per batch and its purity is 99.95 % (Van Der Linde et al., 2000). PRODUCTION OF [15O]OZONE USING A HETEROGENEOUS TARGET Although [15O]O3 has been observed as an impurity in several oxygen-15 production target systems (Dyson et al., 1958; Buckingham & Forse, 1963; Welch & Ter-Pogossian, 1968) the production of high specific activity [15O]ozone suitable for use in life science studies of environmental exposure to ozone has only recently been described. (Dunn et al., 1997). The irradiation of a matrix of silica micro-fibres with protons Ep 27-22MeV has been shown to provide a source of [15O] species which can, to some extent, be reacted with small concentrations of molecular oxygen in an inert diluent gas (Neon/0.5 % O2). Subsequent rapid cryopurification has yielded practicable amounts of [15O]O3 .

CHEMISTRY OF NITROGEN-13 AND OXYGEN-15

131

CHEMICAL CONVERSION OF PRIMARY IN-TARGET PRODUCED PRODUCTS SYNTHESIS OF [15O]H2O As outlined above [15O]H2O can be produced in the target. However, when this is not possible or desirable it can be synthesised by the Pd or Pt catalysed reaction of hydrogen with [15O]O2 (West & Dollery, 1961; Clark & Buckingham, 1975; Berridge et al, 1990b) or by the isotopic exchange of [ I5 O]CO 2 with water (Welch et al, 1969). SYNTHESIS OF [15O]CO2 In-target production of [15O]CO2 has been outlined above, however, where this approach is not possible or desirable [15O]CO2 may be synthesised by the reaction of [15O]O2 with carbon at 40Q-450°C. An metal oxide catalysed isotopic exchange between [15O]O2 and CO 2 has been alternative method based on mi described (Iwataefa/., 1988). SYNTHESIS OF [15O]CO As discussed above the in target production of [15O]CO may occur as a result of the radiolytic processes that occur therein when CO is present. (Welch & Ter-Pogossian, 1968). Attempts have been made to optimise in-target conversion by providing a source of hot carbon within the target volume (Votaw et al,. 1986). The latter method showed a significant potential for reducing the need for O2 carrier in the target gas and hence the resulting stable CO is also reduced, which is of significant concern in its biological application due to toxicity concerns. Similarly when [15O]O2 from the target is reacted with carbon at 900 to 950°C the stable CO content must be carefully controlled. Operation of the target at minimal O2 concentration has been found to provide an effective solution to this problem (Strickmans et al., 1985; Berridge et al., 1990b), SYNTHESIS OF [15O]BUTANOL The reaction of [15O]O2 with tri-n-butylborane in THF has been used to synthesise [l5O]BuOH (Kabalka et al., 1985). Subsequent refinements of this reaction using immobilised tri-n-butylborane on an alumina SepPak* cartridge have been performed. This has enabled the automation of multi-dose preparation systems (Goodmann et al., 1991; Bauer & Wagner, 1991). Low concentrations of carrier O2 are recommended (Berridge et al 1986, Berridge et al., 1990a).

15

o2

THF Scheme 8: Synthesis of 15O-labelled butanol SYNTHESIS OF [15O]HYDROGEN PEROXIDE A method has been described for the preparation of [15O]H2O2 (Takahashi et al., 1989). It is based the published procedure for the preparation of [18O]H2O2 via the [ O ] O 2 autoxidation of the organic

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HANDBOOK OF RADIOPHARMACEUTICALS

reducing agent 2-ethylanthrahydraquinol. The 2-ethylanthrahydraquinol was immobilised on a C-18 SepPak® by evaporation of the solution in ethyl acetate. [I5O]O2 was passed through this pre-loaded SepPak® to produce [15O]H2O2 (Scheme 9). The [15O]H2O2 was then recovered using a 6ml wash of isotonic NaCl solution (0.9 % NaCl in water). Radio-HPLC on a C-18 column together with a degradative chemical analysis confirmed the presence and specific activity of the [15O]H2O2. 15

02

-^-

-^

- H215O2

OH

2-Ethylanthrahydroquinol

2-Ethylanthrahydroquinone

Scheme 9: Synthesis of 15O labelled hydrogen peroxide

QUALITY CONTROL OF 15O LABELLED COMPOUNDS Oxygen-15 labelled compounds are often used in biological studies in man. In order to ensure the safety and efficacy of the compound significant attention has been paid to the implementation of the relevant analytical tools. Radio gas chromatography: Gas phase products labelled with I5O are readily analysed using a Gas Chromatograph (GC) equipped with both a chemical detector and a radioactivity detector. A thermal conductivity detector (Katharometer) is usually the detector of choice together with a positron sensitive radiation detector, although a well shielded Nal(Tl) gamma scintillation counter can also be used (Welch & Ter-Pogossian, 1968; Clark & Buckingham, 1975). Data is usually acquired into a PC based data recorder/integration package with software with peak decay correction capabilities. Automated systems have been described (Strickmanns et al., 1985). The GC analysis method can be based on using two separate injections on different columns for a full analysis, a Molecular Sieve 5A (MS) and a PoraPak Q® (PQ), or using a concentric column which contains an MS inner and PQ outer packing (supplied by Alltech®, type CTR-1). This column permits all the relevant gasses (O2, CO2, N2, CO & N2O) to be conveniently assayed with one injection (Welch & Kilboum, 1985; Strickmanns et al., 1985; Clark et al., 1987). Radio-HPLC: Quality control of the injectable forms of 15O labelled compound [15O]H2O and [15O]BuOH is carried out using Radio-HPLC. A method has been described for [15O]H2O (Metzke et al., 2001) and [15O]BuOH (Bauer & Wagner, 1991; Berridge et al., 1990a).

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133

RADIOTRACER DELIVERY AND WASTE MANAGEMENT SYSTEMS When used for biomedical research applications the gas phase

15

O compounds are delivered either

continuously (steady state methods) or as a bolus (dynamic methods) mixed with air. The essential features of a steady state delivery system involve the regulation of the target beam current, target gas flow and air dilution flow to maintain a constant delivery MBq/sec (mCi/min) as measured by a flow through lonisation Chamber and Mass Flow Controller. Administration of the regulated mixture is usually via a medically acceptable breathing system, e.g., face mask, nasal tubes or ventilator (LeBars et al., 1990; Wagner et al, 1993; Jackson et al, 1993; Lottgen et al, 1994). For the bolus methods generally much higher radioactive concentrations are required to fill a flexible vessel (often a rubber anaesthetic bag) with approximately one full inspirate (breath) of the product diluted in air. In order to measure the radioactivity in this bolus preparation the bag is mounted in a large volume calibrated radiation detector, usually in the form of an ionisation chamber. Administration is usually via a mouthpiece and one-way non re-breathing valve or a ventilator (Wagner et al., 1993). The development of systems for the safe delivery of both bolus and steady state intravenous infusions of [15O]H2O has received a lot of attention. Early methods were based on passing the water vapour, which had been generated by one of the methods described above, through an isotonic solution of NaCl. A sample of this solution would then be drawn up into a syringe prior to injection (Welch et al., 1969; Clark & Buckingham, 1975; Welch & Kilbourn, 1985). With the introduction of the use of PET in functional brain "activation studies", where multiple doses, typically 12 to 16 doses, of [15O]H2O need to be delivered for each subject studied, it became urgent to automate the whole process. Several systems have been described which achieve many of the safety features required to protect both the subject being injected and the operators preparing and administering the radiotracer (Meyer et al., 1984; Clark & Tochon-Danguy, 1992; Ferried et al., 1994; Palmer et al., 1995). The safe disposal of waste patient expired and unused gaseous products depends on the layout of the facilities and the local regulatory requirements. Several systems have been described in the literature (Peters et al, 1991; Wagner et al., 1993; Tochon-Danguy et al., 1994). REFERENCES Barrio JR, Baumgartner FJ, Phelps ME, Henze E, Schelbert HR and MacDonald NS (1982) N-13 Labeled branched-chain L-amino acids. Synthesis and enzyme kinetics. J. Nucl. Med. 23: 107. Bauer B and Wagner R (1991) Improved synthesis of [15O]Butanol for clinical use. J. Label. Compd. Radiopharm. 30: 69–71. Baumgartner FJ, Barrio JR, Henze E, Shelbert HR, MacDonald NS, Phelps ME and Kuhl DE (1981) 13Nlabeled L-amino acids for in vivo assessment of local myocardial metabolism. J. Med, Chem., 24: 764-766. Beaver JE, Finn RD and Hupf HB (1976) A new method for the production of high concentration 15Olabelled carbon dioxide with protons. Int. J. Appl. Radiat. Isot., 27:195–197.

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Berridge MS, Franceschini MP, Tewson TJ and Gould KL (1986) Preparation of oxygen-15 butanol for positron tomography. J. Nucl. Med., 27: 834–837. Berridge MS, Cassidy EH and Terns AH (1990a) A routine, automated synthesis of oxygen-15 labelled butanol for positron tomography. J. Nucl. Med., 31: 1727-1731. Berridge MS, Terris AH and Cassidy EH (1990b) Low-carrier production of water and carbon monoxide. Appl. Radial. Isot., 41: 1173–1175. Berridge MS and Landmeier BJ (1993) In-target production of [N-13]ammonia - target design, products, and operating parameters. Appl. Radial. Isot., 44: 1433–1441. Buckingham PD and Forse GR (1963) The preparation and processing of radioactive gases for clinical use. Int. J. Radial, Isot., 14: 439-445. Chasko JH and Thayer JR (1981) Rapid concentration and purification of N-13-labeled anions on a highperformance anion-exchanger. Int. J. Appl. Radial Isot., 32: 645–649. Clark JC and Buckingham PD (1975) Short Lived Radioactive Gases for Clinical Use. Butterworths, London. Clark JC, Crouzel C, Meyer GJ and Strickmans K (1987) Current methodology for oxygen-15 production for clinical use. Appl. Radial. Isot., 38: 597–600. Clark JC and Tochon-Danguy H (1992) R2D2, a bedside [oxygen-15] water infuser. PSI Proceedings 9201, pp234-235 ISSN 1019-6447. (Proc. IVth Int. Workshop on Targetry and Target Chemistry PSI Villigen , Switzerland 1991). Cohen MB, Spoiler L, Chang CC, MacDonald NS, Takahashi J and Bobinet DD (1974) Immobilzed enzymes in the production of radiopharmaceutically pure amino acids labeled with 13N. J. Nucl. Med., 15: 1192–1195. Cooper AJ and Gelbard AS (1981) The use of immobilized glutamate dehydrogenase to synthesize I3 Nlabeled L-amino acids. Anal. Biochem., 111: 42–48. Cooper AJL, Gelbard AS and Freed BR (1985) Nitrogen-13 as a biochemical tracer. Adv. Enzymol. Relat. Area in Mol Biology. 57: 251-356. De Spiegeleer B, Sieger G, Vandecasteele C, Van den Bosche W, Schelstraete K, Claeys A and De Moerloose P (1986) Microscale synthesis of nitrogen-13 labeled cisplatin. J. Nucl. Med., 27: 399-403. Dence CS, Welch MJ, Hugehy B, Shefer RE and Klinkowstein RE (1994) Production of [N-13]ammonia applicable to low energy accelerators. Nucl. Med. Bioi, 21: 987-996. Digenis GA, Cheng YC, McQuinn RL, Freed BR and Tibury RS (1981) N-13 labelling of a substituted nitrosourea, its carbamate, and nitrosocarbonyl - in vivo and in vitro studies. Short-lived Radiopharmaceuticals in Chemistry and Biology, Advances in Chemistry Series 197, Root JW and Krohn KA, ACS, Washington DC, pp 351–367. Digenis GA, McQuinn RL, Freed B, Tilbury RS, Reiman RE and Cheng YC (1979) Preparations of 13Nlabeled streptozotocin and nitrosocarbaryl. J. Label. Compd. Radiopharm., 16: 95-%. Diksic M, Yamamoto YL and Feindel W (1983) An on-line synthesis of [I5O]N2O: New blood flow tracer for PET imaging. J. Nucl. Med., 24: 603-607. DolleryCTand West JB (1960) Metabolism of oxygen-15. Nature 189: 1121.

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Dunn WL, Russell ML, Weiland BW and Yacout AM (1997) Monte carlo analysis of multi-layer targets for production of 15O. Appl. Radiat. Isot., 48: 1591–1600. Dyson NA, Hugh-Jones P, Newbery GR and West JB (1958) Preparation and use of oxygen-15 with particular reference to its value in study of pulmonary malfunction, pp 105–115. Proceedings of the second UNESCO International conference on Peaceful Uses of Atomic Energy Geneva, Pergamon Press. Dyson NA, Sinclair JD and West JB (I960) A comparison of the uptakes of oxygen-15 and oxygen-16 in the lung. J. Physiol, 152: 325–336. Elmaleh DR, Hnatowitch DJ and Kulprathipanja S (1979) A novel synthesis of 13N-L-asparagine. J. Label. Compd. Radiopharm., 16: 92-93. Feigl F and Oesper R (1972) Spot Tests in Inorganic Analysis. Elsevier, Amsterdam. Ferneri RA, Schyler DJ, Wieland BW and Wolf AP (1983) On-line production of [N-13]-nitrogen from solid enriched [C-13]-targets and it's application to [N-13]ammonia using microwave radiation. Int. J, Appl. Radiat. Isot., 34: 897–900. Ferried RA and Wolf AP (1983) The chemistry of positron-emitting nucleogenic (hot) atoms with regard to preparation of labelled compounds of practical utility. Radiochimica Acta 34: 69-83. Ferrieri RA, Alexoff DL, Schlyer DJ and Wolf AP (1994) Remote processing, delivery and injection of H2[15O] produced from a N2/H2 gas target using a simple and compact apparatus. Appl. Radiat. Isot. ,45: 1149–1154. Finn RD, Christman DR and Wolf AP (1981) A rapid synthesis of nitrogen-13 labeled amphetamine. J. Label. Compd. Radiopharm., 18: 909–913. Firouzbakht ML, Schlyer DJ, Wolf AP and Fowler JS (1999) Mechanism of nitrogen- 13-labeled ammonia formation in a cryogenic water target. Nucl. Med. Biol., 26: 437–441. Fowler JS and Wolf AP (1986) Positron emitter-labeled compounds: Priorities and problems. In [Positron Emission Tomography and Autoradiography: Principles and Application for the Heart and Brain], Phelps ME, Mazziota JC and Schelbert HR (Eds), Raven Press, New York. pp 391–450. Gatley SJ and Shea C (1991) Radiochemical and chemical quality-assurance methods for the [13N]ammonia made from a small volume H216O target. Appl. Radiat. Isot., 42: 793-796. Gelbard AS, Carke LP and Laughlin JS (1974) Enzymatic synthesis and use of 13N-labeled L-asparagine for myocardinal imaging. J. Nucl. Med., 15: 1223-1225. Gelbard AS, Carke LP, MacDonald JM, Monahan W, Tilbury RS, Kuo TYT and Laughlin JS (1975) Enzymatic synthesis with 13N-labeled L-glutamine and L-glutamic acid. Radiology 116: 127132. Gelbard AS and Cooper AJL (1979) Synthesis of (13N)-labeled aromatic L-amino acids by enzymatic transamination of (l3N)-L-glutamic acid. J. Label. Compd. Radiopharm., 16; 94. Gelbard AS, Cooper AJL, Asano Y, Nieves E, Filc-Dericco S and Rosenspire KC (1990) Method for the enzymatic synthesis of tyrosine and phenylalanine labeled with nitrogen-13. Appl. Radiat. Isot., 41: 229-233.

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Goodman MM, Devinney JL, Kabalka GW, Longford CPD, Ladesky M and Green JF (1991) Computer controlled synthesis of oxygen-15 butanol and water automated production and dispensing systems. J. Label, Compd. Radiopharm., 30: 166–168. Hagami E, Murakami M, Takahashi K, Kanno I, Aizawa Y, Hachiya T, Shoji Y, Shishido F and Uemura K (1986) Studies on the direct synthesis of [O-15]-H2O. Nuclear Medicine 23: 351-358. (Japanese) Harper PV and Wickland T J (1981) Oxygen-15 labelled water for continuous administration J. Label. Compd. Radiopharm., 18: 186. Herscovitch P, Raichle ME, Kilbourn MR and Welch MJ (1987) Positron emission tomography measurment of cerebral blood flow and permeability-surface area product of water using I5Owater and 11C-butanol. J. Cerebr. Blood Flow 7: 527-542. Holschbach M, Hamkens W, Steinbach A, Hamacher K and Stocklin G (1996) [l3N]Cisplatin: a fast and efficient on-line synthesis using a solid state support. Appl. Radial, hot., 48: 739-744. Hunter WW and Monahan WG (1971) [N–13]Ammonia: a new physiologic radiotracer for molecular medicine. J. Nucl. Med., 12: 368. Ido T and Iwata R (1981) Fully automated synthesis of 13NH3.J. Label. Compd. Radiopharm., 18: 244246. Iwata R, Ido T, Fujisawa Y and Yamazaki S (1988) On-line interconversion of [15O]O2 and [15O]CO2 via metal oxide by isotopic exchange. Int. J. Appl. Radiat, Isot., 39: 1207–1211. Jackson JR, Dembowski BS, Erenkaufer RL, Mclntyre E and Reivich M (1993) [15O]H2O, [15O]O2 and [15O]CO gas production monitoring and control system. Appl. Radiat. hot., 44: 631–634. Joliot F and Curie I (1934) Artificial production of a new kind of radio-element. Nature 133: 201–202. Kabalka GW, Lambrecht RM, Sajjad M, Fowler JS, Kunda SA, McCollum GW and Macgregor R (1985) Synthesis of [l5O]-labelled butanol via organoborane chemistry. Int. J. Appl. Radiat. Isot., 36: 853-855. Kabalka GW, Wang Z, Green JF and Goodman MM (1992) Synthesis of isomerically pure nitrogen-13 labeled gamma-aminobutyric acid and putrescine. Appl. Radiat. hot., 43: 389–391. Kabalka GW, Goodman MM, Green JF, Marks R and Longford D (1993) Synthesis of nitrogen-13 labelled amines using organoborane polymers. J. Label. Compd. Radiopharm., 32: 165. Kamen MD (1957) The isotopes of oxygen, nitrogen, phosphorous and sulfur. In Isotopic Tracers in Biology: An Introduction to Tracer Methodology. Orlando Fla. Academic Press, page 339 Kaseman DS, Cooper AJL and Meister A (1984) Synthesis of hydroxyl[N-13]amine and binding 13 NH2OH to 2 transaminases. J. Label. Compd. Radiopharm., 21: 803–814. Kiso Y, linuma S, Mimoto T, Saiji H, Yokoyama and Akaji K (1991) A synthetic method for the rapid preparation of 13N-labeled dermorphin analogue, H-Tyr-D-Met(O)-Phe-Gly-NH2 (SD-62). Chem. Pharm. Bull (Tokyo) 39: 2734-2736. Kothari PJ, Finn RD, Kabalka GW, Vora MM, Boothe TE, and Emran AM (1986) Synthesis of N-13 labeled alkylamines via amination of organoboranes. Appl. Radiat. Isot., 37: 469-470. Krasikova RN, Fedorova OS, Korsakov MV, Bennington Bl and Berridge MS (1999) Improved [N13]ammonia yield from the proton irradiation of water using methane gas. Appl. Radiat. hot., 51:395v401.

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Krizek H, Lernbares N, Dinwoodie R and Harper PV (1973) Production of radiochemically pure 13NH3 for biomedical studies using the 16O(p,a)13N reaction. J. Nud, Med., 14: 629–630. Krizek H, Harper PV, and Mock B (1977) Adapting the old to new needs. 13N-labeled urea. J. Label. Compd. Radiopharm., 13: 207. Krohn KA, Link JM, Lewellen TK, Risler R, Eenmaa J and Maier M (1986) The use of 50 MeV protons to produce carbon-11 and oxygen-15. J. Label. Compd. Radiopharm. 23: 1190–1192. Lade RE, Castigloni M and Wolf AP (1979) ACS Meeting, Washington DC. Landais P, Waltz P, Tochon-Danguy H, Goethals P, Strijckrnans K, Rose K and Offord RE (1993) Preparation of nitrogen-13 labelled polypeptides for positron emission tomography. J. Label. Compd. Radiopharm., 32: 171. Le Bars D, La Venne F, Sasse K, Landais P and Cinotti L (1991) Development of an inexpensive programmable logic controller for clinical gases regulation, /. Label. Compd. Radiopharm., 30:113 Le Bars D (2001) A convenient production of [13N]nitrogen for ventilation studies using a nitrogen gas target for UC production. J. Label. Compd. Radiopharm., 44: 1–5. Lenzi GL, Jones T, McKenzie CG, Buckingham PD, Clark JC and Moss S (1978) Study of regional cerebral metabolism and blood flow relationships in man using the method of continuously inhaling oxygen-15 and oxygen-15 labelled carbon dioxide. J. of Neurolog., Neurosurg. and Psychiatry 41:1–10. Lindner L, Helmer J and Brinkman GA (1979) Water "loop"-target for the in-cyclotron production of 13N by the reaction 16O(p,a)13N. Int. J. Appl. Radiat. Isot., 30: 506-507. Lottgen J, Wagner R, Richerzhagen N and Wienhard K (1994) Automatic control device for the continuous administration of 15O labelled gaseous tracers for PET measurements. Appl. Radiat. Isot., 45: 923–928. Lunn RJ (1995) Inhaled nitric oxide therapy. Mayo Clin. Proc., 70: 247–225. Majumder C, Lathrop KA, and Harper PV (1977) Synthesis and analysis of 13N-asparagine for myocardial scanning. J. Label. Compd. Radiopharm., 13: 206. Metzke K-H, Meyer GJ and Knapp WH (2001) Fast quality control of radiopharmaceutical by integrated recovery measurement in radio-HPLC. J. Label. Compd. Radiopharm., 44: S835-S837. McCarthy TJ, Dence CS, Holmberg SW and Welch MJ (1996) Inhaled [N-13]nitric oxide: A positron emission tomography (PET) study. Nud. Med. BioL, 23: 773–777. McElfresh MW, Meeks JC and Parks NJ (1979) The synthesis of 13N-labelled nitrite of high specific activity and purity. J. Radioanal. Chem., 53: 337–344. Meeks JC, Wolk CP, Lockau W, Schilling N, Shaffer PW and Chien WS (1978) Pathways of assimilation of [ I3 N]N 2 and 13NH4+ by cyanobacteria with and without heterocysts., J Bacterial., 134: 125-30. Meyer GJ, Schober O, Bossaller C, Sturm J and Hundershagen H (1984) Quantification of regional l5Olabelled water. Eur. J. Nucl. Med., 9: 220-228. Mtiller GO (1967) Praktikum der quantitativen chemischem Analyse. S. Hitzel Verlag, Leipzig, pp 254– 256.

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Mullholland GK, Sutorik A, Jewett DM, Manger JJ and Kilbourn MR (1989) Direct in-target synthesis of [I3N]NH3 by irradiation of water under hydrogen pressure. J. Nucl. Med., 30: 926. Mulholland GK, Kilbourn MR and Moskwa JJ (1990) Direct simultaneous production of 15O water and 13 N ammonia or 18F fluoride ion by 26 MeV irradiation of a double chamber water target. Appl. Radiat. Isot., 41: 1193–1199. Nickles RJ, Gatley SJ, Hichwa RD, Simpkin DJ and Martin JL (1978) The synthesis of nitrogen-13 nitrous oxide. Int. J Appl. Radiat. Isot., 29: 225–227. Nicholas DJD, Silvester DJ and Fowler JF (1961) Use of radioactive nitrogen in studying nitrogen fixation in bacterial cells and their extracts. Nature 189: 634-636. Nieves E, Rosenspire KC, Filc-DeRicco S and Gelbard AS (1986) High-perfomance liquid chromatographic on-line flow-through radioactivity detection system for analysing amino acids and metabolites labeled with nitrogen-13. J. Chromatogr 383: 325-337. Palmer BM, Sajjad M and Rottenberg DA (1995) An automated [15O]H2O production and injection system for PET imaging. Nucl. Med. Biol., 22: 214–249. Parks NJ and Krohn KA (1978) The synthesis of 13N labelled ammonia, dintrogen, nitrite and nitrate using a single cyclotron target system. Int. J. Appl. Radiat. Isot., 29: 754–756. Peters JM, Quaglia L, Delfiore G, Hannay J and Fissore A (1991) A system for the disposal of large volumes of air containing O-15. Nucl. Instrum. Methods Phys Res Section A-Accelerators Spectrometers Detectors and Associated Equipment. 300(N2): 409 Pettit WA, Mortara RH, Digenis GA and Reed MF (1975) Preparation of nitroso-13N-nitrosoureas. J. Med. Chem., 18: 1029–1031. Pettit WA, Tilbury RS, Digenis GA and Mortara RH (1977) A convenient synthesis of 13 N-BCNU. J. Label. Compd. Radiopharm., 13: 119–122. Phelps ME, Hoffman EJ and Rayboud C (1977) Factors which affect cerebral uptake and retention of N-13 NH3. Stroke 8: 694-702. Root JW (1981) Advances in Chemistry Series, 197, ACS, Washington DC. Ruben S, Hassid WZ and Kamen MD (1940) Radioactive nitrogen in the study of N2 fixation by leguminous plants. Science 91: 578. Ruth TJ (1985) The production of [18F]F2 and [!5O]O2 sequentially from the same target chamber. Int. J. Appl. Radiat. Isot., 36:107–110. Sajjad M, Lambrecht RM and Wolf AP (1984) Cyclotron isotopes and radiopharmaceuticals XXXIV. excitation function for the 15N(p,n)15O reaction. Radiochimica Acta 36: 159-162. Sajjad M, Lambrecht RM and Wolf AP (1985) Cyclotron isotopes and radiopharmaceuticals XXXVI. investigation of some excitation functions for the preparation of I5O. !3N and 11C. Radiochimica Acta 38: 57–63. Schelbert HR (1986) PET studies of the heart. In Positron Emission Tomography and Autoradiography: Principles and Application for the Heart and Brain, Phelps ME, Mazziota JC and Schelbert HR (Eds), Raven Press, New York, pp 581–662. Schuster DP (1998) The evaluation of lung function with PET. Semin. Nucl. Med., 28: 341–351. Segal AW, Clark JC, and Allison AC (1978) Tracing the fate of oxygen consumed during phagocytosis by human neutrophils with I5 O 2 Clin. Sci. Mol. Med., 55: 413–415.

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Siri WE (1949) Major organic metabolites. In Isotope Tracers and Nuclear Radiations With Applications to Biology and Medicine. McGraw-Hill pp 157. Straatman MG and Welch MJ (1973) Enzymatic synthesis of nitrogen-13 labeled amino-acids. Radiat. Res., 56: 48–56. Straatman MG (1977) A look at 13N and 15O in radiopharmaceuticals. Int. J. Appl. Radiat. Isot., 28: 13– 20. Strickmans K, Vandecasteele C and Sambre J (1985) Production and quality control of 15O2 and C15C>2 for medical use. Int. J. Appl. Radiat. Isot., 36: 279-283. Suzuki K, Iwata R, Tamate K, Yoshikawa K and Ido T (1977) Remote system for the mass production of shortlived radioisotopes in a cyclotron. Development of the system and radioisotope production. Radioisotopes 26: 67-73. Suzuki K, Tamate K, Nakayama T, Yamazaki T, Kasida Y, Fukushi K, Maruyama Y, Maekawa H and Nakaoka H (1982) Development of an equipment for the automatic production of 13NH3 and L( l3 N)-glutamate.y. Label. Compd. Radiopharm,, 19: 1374-1375. Suzuki K, Yoshida Y, Shikano N and Kubodera A (1999) Development of an automated system for the quick production of N-13-labled compounds with high specific activity using anhydrous [N13]NH3. Appl. Radiat. Isot., 50: 1033–1038. Takahashi K, Murakami M, Hagami E, Sasaki H, Kondo Y, Mizusawa S, Nakamichi H, lida H, Miura S, Kanno I and Uemura K (1989) Radiosynthesis of 15O-labelled hydrogen peroxide. J. Label. Cmpd. Radiopharm., 27:1167–1175. Ter-Pogossian and Powers WE (1958) The use of radioactive oxygen-15 in the determination of oxygen content in malignant neoplasms. In Radioisotopes in Scientific Research, Vol 3 pages 1-11, Proceedings of First UNSECO International Conference, Paris. Pergamon. Ter-Pogossian MM and Herscovitch P (1985) Radioactive oxygen-15 in the study of cerebral blood flow, blood volume and oxygen metabolism. Semin. Nucl. Med. 15: 377–394. Ter-Pogossian MM, Spratt JS, Rudman S and Spencer A (1961) Radioactive oxygen-15 in the study of kinetics of respiration. Am. J. of Physiol., 201: 582-586. Thomas J, Meeks JC, Wolk CP, Shaffer PW and Austin SM (1977) Formation of glutamine from [ l3 N]ammonia, [13 N]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrical. Bacterial., 129: 1545-55. Tilbury RS, Dahl JR, Monahan WG and Laughlin JS (1971) The production of [13N]ammonia for medical use. Radiochem. Radioanalyt. Lett., 8: 317–323. Tibury RS and Dahl JR (1979) I3N species formed by proton irradiation of water. Radiat. Res., 79: 2223. Tochon-Danguy HJ, Midgley SM, Egan GF Sachinidis JI, Phan KS and Chan JG Monitoring and disposal system for cyclotron produced short-lived gases (1994) J. Label. Compd. Radiopharm., 35:601–602. Tominaga T. Inoue O, Irie T, Suzuki K, Yamasaki T and Hirobe M (1985) Preparation of N-13-betaphenethylamine. Int. J. Appl. Radiat. Isot., 36: 555–560. Tominaga T, Inoue O, Suzuki K, Yamasaki T and Hirobe M (1986) Synthesis of 13N-labeled amine by reduction of !3N-labeled amides. Int. J. Appl. Radiat. Isot., 37: 1209–1212.

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Vaalburg W, Kamphusi JAA, Beerling-van der Moles HD and Woldring MG (1975) An improved method for the production of 13N-labeled ammonia. Int. J. Appl. Radiat Isot., 26: 316–318. Vaalburg W, Steenhoek A, Paans AMJ, Peset R, Reiffers S and Woldring MG (1981) Production of I3 Nlabeled molecular nitrogen for pulmonary-function studies. J. Label. Compd. Radiopharm., 18: 303-308. Van Der Linde SC, Jansen WPA, De Goeij JJM, Van Ijzendoorn LJ and Kapteijn F (2000) In-target production of high specific radioactivity [15O] nitrous oxide by deuteron irradiation of nitrogen gas. Appl. Radiat. Isot., 52: 77–85. Vaverek MT and Mulholland GK (1995) Simple general synthesis of NCA [l3N]nitrosothiols and [13N]nitrosamines. J. Label. Compd. Radiopharm., 37: 118–119. Vera Ruiz H and Wolf AP (1977) Excitation function for I5O production via the l4N(d,n)15O reaction. Radiochimica Acta. 24: 65–67. Vera Ruiz H and Wolf AP (1978) Direct synthesis of 15O-labelled water at high specific activities. J. Label. Compd. Radiopharm., 15: 185-189. Votaw JR, Satter MR, Sunderland JJ, Martin CC and Nickles RJ (1986) The Edison Lamp: [15O] carbon monoxide production in the target. J. Label. Compd. Radiopharm., 23: 1211–1213 Wagner R, Arenz W, Richerzhagen N and Wienhard K (1993) Single-breath inhalation or rebreathing of gases labelled with positron emitters: Some technical aspects of dispensing and waste gas management. Appl. Radiat. Isot., 44: 1065-1068. Welch MJ and Ter Pogossian (1968) Preparation of short half-lived radioactive gasses for medical studies. Radiation Research 36: 580-587. Welch MJ, Lifton JF and Ter Pogossian MM (1969) Preparation of millicurie quantities of oxygen-15 labelled water. J. Label. Compd. Radiopharm., 5: 168–172. Welch MJ and Lifton JF (1971) The fate of nitrogen-13 formed by the 12C(d,n)13N reaction in inorganic carbides. J. Am. Chem. Soc., 93: 3385–3388. Welch MJ and Straatmann MG (1973) The reactions of recoil I3N atoms with some organic compounds in the solid and liquid phases. Radiochimica Acta. 20: 124–129. Welch MJ and Kilbourn MR (1985) A remote system for the routine production of I5 Oradiopharmaceuticals. J. Label. Compd. Radiopharm., 22: 1193–1200. West JB and Dollery CT (1961) Absorption of inhaled radioactive water vapour. Nature. 189: 588. Wieland B, Schmidt DG, Bida G, Ruth TJ and Hendry GO (1986) Efficient, economical production of oxygen-15 labelled tracers with low energy protons. J. Label. Compd. Radiopharm., 23: 1214–1216. Wieland BW, Bida G, Padgett H, Hendry G, Zippi E, Kabalka G, Morelle JL, Verbruggen R and Ghyoot M (1991) In-target production of 13N-ammonia via proton irradiation of dilute aqueous ethanol and acetic acid mixtures. Appl. Radiat. Isot., 42: 1095–1098. Wieneke J and Nebeling B (1990) Improved methods for the 13N-application in the short term studies on NO3 fluxes in barley and squash plants. Z. Pfanzenerna.hr. Bodenk. 153: 11–123. Wijns W and Camici PG (1997) The value of quantitative myocardinal perfusion imaging with positron emission tomography in coronary artery disease. Herz.., 2: 87. Whitehead AB and Foster JS (1958) Nuclear cross section for the 16O(p,a)13N reaction. Can. J. Phys., 36: 1276. Wolk CP, Thomas J, Shaffer PW, Austin SM and Galonsky A (1976) Pathway of nitrogen metabolism after fixation of 13N-labeled nitrogen gas by the cyanobacterium, Anabaena cylindrical. J. Biol. Chem., 25: 5027-5034.

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C-LABELLED

GUNNAR ANTONI,1 TOR KIHLBERG12 AND BENGT LANGSTROM12 Uppsala University PET Centre,1 Institute of Chemistry? Uppsala University S-751 85 Uppsala, Sweden INTRODUCTION HISTORY AND BACKGROUND Crane and Lauritsen made the first production of the short-lived positron emitting radionuclide 11C in 1934 (Crane & Lauritsen, 1934). They investigated the physical properties of this radionuclide and demonstrated that 11C decays by positron emission to the stable nuclide 11B. Due to its favourable decay characteristics (t1/2=20.4 min, 98.1% by P+ emission, 0.19% by electron capture) it was considered interesting as a labelling tool for medical purposes. The potential of obtaining 11C with high specific radioactivity was an important factor contributing to the early application of UC in tracer studies. Another consequence of the high specific radioactivity was that the decay products could be disregarded with respect to any biological relevance, an important aspect when considering the use of 11C-labelled compounds in humans. The first biological application employing 1!C was performed by Ruben in 1939 who investigated photosynthesis in plants using [11C]carbon dioxide (Ruben et al., 1939). The potential of 11C-labelled compounds for non-invasive probing of physiological and biochemical processes in humans was then subsequently realized. The first 11C experiment on humans was performed by Tobias in 1945 who studied the fixation of [11C]carbon monoxide by red blood cells (Tobias et al., 1945). In an early phase of research with 11C-compounds, the need for particle accelerators for radionuclide production and the difficulties encountered during 11C-labelling synthesis restricted the development of this research area. When the reactor-produced long-lived isotope 14C (t1/2= 5730y) became available during the Second World War interest shifted towards biological applications with this radionuclide. An advantage of 11C, compared to other positron emitting radionuclides, was that the half-life allowed both repeated PET studies in the same subject within a relatively short time frame, and multi-step syntheses to be performed. In 11C-labelling synthesis a stable carbon is substituted with the radioactive isotope 11C. The 11Clabelled compound obtained is indistinguishable from the stable natural counterpart with respect to biological properties. Theoretically a kinetic isotope effect may occur but this is usually unmeasurable within a complex biological setting and is thus usually disregarded. The possibility to measure a kinetic isotope effect for example, 11C/14C, in a specific chemical reaction, has been of interest from a mechanistic point of view (Axelsson et al., 1987, 1992). The main application of C-compounds are as tracers in biomedical research. The combination of 'C-tracers and positron emission tomography has also proved to be an important diagnostic tool in clinical applications. Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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The development of methods for the synthesis of 11C-compounds to be used as tracers in biomedical research is important for the future progress of the imaging technique Positron Emission Tomography (PET) (Langstrom et al., 1999). This chapter addresses developments of 11C-chemistry with the main focus on achievements presented during the last two decades. For a comprehensive review with references to the earlier work in this field of research see Fowler and Wolf (Fowler & Wolf, 1986). LABELLING STRATEGIES TRACER PRODUCTION Technical considerations Synthetic work with 11C is restricted by the short physical half-life of this radionuclide. The kinetic aspect thus needs attention in selecting a synthetic strategy and rapid reactions are essential. The significance of time as a reaction parameter of equal importance as chemical yield has to be considered in the planning of a labelling synthesis. Since the radiochemical yield is a function of chemical yield and radioactive decay, the maximum radiochemical yield is attained before the reaction has proceeded to completion. This relation between time and concentration of reactants with respect to kinetics is described in some of the initial works on 11C chemistry (Langstrom & Bergson, 1980; Langstrom et al., 198la; Berger et al., 198la). Some of the technical approaches needed when working with high levels of radioactivity with short-lived radionuclides are as follows: * Remote or processor-controlled automated devices The use of remote or processor-controlled automated devices, mainly developed for radiation protection purposes, has other advantages such as increasing the reproducibility in the tracer production. This also facilitates work towards standardized procedures that is an important aspect with respect to quality assurance and the implementation of GMP in PET related work. * One-pot and on-line procedures One-pot and on-line procedures are other important means to facilitate the technical handling. * Miniaturization Miniaturization of synthesis equipment gives the option to reduce the amount of reagents, which may be advantageous for product purification and for the increase of the specific radioactivity. * On-column preparations and closed loops The use of on-column preparations and closed loops as supports for the reaction are other technical solutions that are valuable complements to performing the reaction in a small reaction vial (Jewett et al., 1985; Iwata et al., 1992). * Sonication and microwaves Sonication (Le Breton et al., 1991) or microwaves (Niisawa et al., 1984; Hwang et al., 1987; Stone-Elander et al., 1994; Thorell et al., 1992) can be used to speed-up the reaction. The balance between product formation and decomposition can thus be favourable due to a short reaction time, although drastic reaction conditions may be used. *

Salvation parameters

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Extended control of the solvation parameters by performing the synthesis in a supercritical media may also offer some advantages (Jacobson et al., 1994a, b, 1995, 1996). Synthetic considerations For the production of 11C-labelled tracers four main approaches have been employed: * *

Biosynthetic methods Recoil labelling

* *

Organic synthetic methods Enzyme catalysis

Biosynthetic methods using plant leaves or algae (Lifton & Welch, 1971) and recoil labelling (e.g., the spallation reaction l6O(p,3p3n)11C) are of low importance. Hence, of the labelling approaches organic synthesis and enzymatic catalysis are the only methods of practical interest. The development of rapid labelling syntheses is highly dependent on the availability of suitable labelled precursors. Only a few useful target-produced primary precursors are available such as [11C]carbon dioxide and [11C]methane. Therefore, much work has been devoted to the development of methods for routine production of secondary precursors to be used as building blocks in the production of useful PET tracers. Synthetic strategies that give access to a variety of labelling positions are thus essential for the further development of the tracer applications in PET. A convergent synthetic approach should preferable be used, i.e., the synthesis should be designed in such a way that the 11C atom is introduced as late as possible in the synthesis in order to decrease synthesis time and thus optimize the uncorrected radiochemical yield. This approach is exemplified by alkylations of carbon, nitrogen, oxygen and sulphur nucleophiles with methyl iodide, a synthetic route that provides a general method for labelling many biomedically interesting compounds. * Position specific labelling Most biomolecules contain several carbon atoms, which give the possibility to select the position to label in the molecule (i.e., position-specific labelling). Since hetero atom bound methyl groups are common in many biomolecules and pharmaceuticals, the introduction of [11C]methyl iodide as a generally useful alkylating agent opened up new perspectives in labelling synthesis. Another general synthetic approach that was adopted was to label in the carbonyl position using [11C]carbon monoxide or [uC]phosgene. This has a primary value for PET tracer synthesis since biologically active substances often contain carbonyl groups or functionalities that can be derived from a carbonyl group. *

Kinetics

The stoichiometric relationships in labelling synthesis with radionuclides of high specific radioactivity need special attention. The alkylation of amines with methyl iodide is characterized by product mixtures with quartenarized amines as the main or end product. This is due to the increased nucleophilicity created from the primary to the secondary and tertiary amines. However, in 11C-labelling synthesis the amount of [11C]methyl iodide is very small and the excess of the amine can be at least 25-100 times. Due to the resultant change from second order to pseudo-first order kinetics, the problem with polyalkylation is minimized and the monoalkylated product obtained.

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* Future perspectives A future perspective in labelling synthesis is focused on organometallic chemistry. Some of the newly introduced technical improvements combined with transition metal catalyst will provide new options that should give rise to increased flexibility in the choice of labelling position and enable the syntheses to be performed within the time-frame set by the short half-life of 11C. Specific radioactivity The theoretical specific radioactivity of 11C is 3.4xl05 GBq/|imol, a limit that is considerably higher than the value of 500-2000 GBq/u,mol, which is present practically attainable at end of radionuclide production (Harada & Hayashi, 1993). * Isotopic dilution - radionuclide production It is practically impossible to completely avoid isotopic dilution with stable carbon isotopes from the target material (e.g., nitrogen/oxygen, nitrogen/hydrogen gas mixtures) and target holder, usually aluminium (Heselius et al., 1987). This means that in the perspective of a 11C tracer synthesis a considerable isotopic dilution by stable carbon is obtained during the radionuclide production. The amount of carbon isolated in the form of carbon dioxide following radionuclide production is typically in the order of 1-25 nmol. * Isotopic dilution - labelling synthesis The degree of isotopic dilution during production of a PET tracer is also determined by the choice of primary precursor and the quality of the reagents used in the chemical transformations. Chemicals such as lithium aluminium hydride and Grignard reagents are usually contaminated by products formed from reaction with atmospheric carbon dioxide which is another source of stable carbon decreasing the specific radioactivity (Iwata, et al., 1988). At the end of a synthesis the amount of the 11C-labelled compound generally is in the range from 10 nmol to 100 nmol. BIOLOGICAL PERSPECTIVES The tracer signal in the biological target should increase to a significant level above the non-specific binding during the time interval of the PET investigation. A tracer should be designed in such a way that it can probe a specific function within the organ of interest. The transport of the compound over biological membranes is thus of importance. Therefore, the pro-drug concept, i.e., a compound that after metabolism in vivo releases the active drug, is an option when the transport over biological barriers restricts site-specific delivery. The label is ideally in a metabolically stable position. This is, however, not always possible to achieve. The fate of the label as a result of metabolism must be considered in selecting the position to label within a molecule. One of the requirements for measuring any biochemical reaction is that the reaction products are known. Various strategies have been adopted to accomplish this, where metabolic trapping has proved to be a useful approach. Metabolite correction has to be taken into account if significant metabolism of the labelled molecule can be envisaged during the course of the study. Selection of a labelling position that gives rise to labelled metabolites that do not interfere with the measurement of the biological process to be studied is another option. This may be achieved by labelling a position that produces a labelled metabolite that lacks selectivity for, and is rapidly cleared from the target organ.

ASPECTS ON THE SYNTHESIS OF 11 C-LABELLED COMPOUNDS

145

Using chiral tracers, high enantiomeric purity is important since the inactive enantiomer becomes a disturbing background source of radioactivity that interferes with the interpretation of the signal from the active enantiomer. However, the inactive enantiomer can be used separately, i.e., to subtract the unspecific binding and verify that the interaction between the biological system and the tracer is specific, METHODS FOR 11C-SYNTHESES LABELLED PRECURSORS Primary precursors - target produced one-carbon compounds Labelled precursors are usually small reactive molecules used as building blocks for the labelling of different structural elements in the target molecule. They can be divided into primary or secondary precursors. Primary precursors are prepared in the target and secondary ones from a primary (usually by on-line or one-pot procedures). The choice of precursor may have an impact on the specific radioactivity of the final product. The best option for optimization of the specific radioactivity is to prepare a molecule in the target that is not present in the atmosphere nor is an impurity in any of the chemicals used in the further processing to the labelled tracer. The in-target production of labelled precursors is practically restricted to [1!C]carbon dioxide and [1!C]methane (Christman et al, 1975). [11C]Carbon dioxide is the most commonly prepared primary precursor. It is usually produced by the 14N(p, oQ)11C reaction from nitrogen gas containing trace amounts of oxygen (Bida et al., 1978). Some [11C]carbon monoxide, another versatile precursor, is formed simultaneously. Although this impurity is easy to separate cryogenically from [11C]carbon dioxide, the yield of the latter can be optimized by passing the target gas over heated CuO and thus convert CO to CO^. [11C]Carbon monoxide production is favoured by increasing the oxygen concentration in the target from ppm level to a few per cent. The main obstacle to this method, apart from lower yield as compared with carbon dioxide and methane production, is the difficulty encountered when separating labelled carbon monoxide from the target gas containing labelled side products. Irradiating a nitrogen/hydrogen gas mixture produces [11C]methane. Hydrogen [11C]cyanide (Lamb et al., 1971) can also be obtained directly in the target by a recoil synthesis using the same gas mixture. Due to radiolysis [11C]methane is the main isolated product when production parameters are set to give enough radioactivity for further synthetic work. An advantage of using target produced [11C]methane is low isotopic dilution, due to a low concentration of methane in the atmosphere and in the chemicals used for further processing. [11C]Cyanamide (Iwata & Ido, 1983) and [11C]guanidine (Iwata et al., 1981) are other examples of carbon-nitrogen precursors prepared in the target. One of the most important labelled precursors, [11C]methyl iodide, can be prepared in the target by a recoil synthesis (Wagner et al., 1981), but the yield is not high enough to be useful. In conclusion [11C]carbon dioxide is the most versatile primary precursor with respect to production yield, ease of separation from target gas and prospect for further useful transformations. Furthermore, of the primary precursors only [11C]carbon dioxide has been used directly as a labelling agent to any great extent. It can be used as a

146

HANDBOOK OF RADIOPHARMACEUTICALS

substitute for phosgene in the formation of ureas (Schirbel et al., 1999) or in reactions with lithium alkyl compounds and Grignard reagents. Secondary precursors The limited access to labelled precursors is a restriction in the choice of reaction route. Development of new secondary precursors is, thus, of importance with respect to the possibility to increase the labelling potential. Several reactive molecules have been prepared by simple on-line or one-pot procedures from [11C]carbon dioxide. Some of the most important secondary precursors obtained from [11C]CO2 are shown in Figure 1.

R"CH

2

OH

fc-

R " CH

2

i

Figure 1. Examples of secondary precursors obtained from [11 C]CO2 ["CJMethane, the other primary precursor, is also a useful source for the preparation of more reactive labelling reagents. 1 1

C w

H 3 o i1 -^« « n

C H C I3

1 1

t

C H

4

—»-1

1

•^ ^^

ii n

C H 2N 1 n

r* fi L/ IN

4

C O C \2

Figure 2. Examples of secondary precursors prepared from [UC] methane So far, [uC]methyl iodide has been the most versatile of these secondary precursors. It can be prepared by reduction of [11C]carbon dioxide with lithium aluminium hydride followed by reaction with hydroiodic acid (see scheme 1) (Langstrom & Lundqvist, 1976; Crouzel et al., 1987a). This method is very reliable, however, suffers from the drawback that the lithium aluminium hydride contains carrier carbon that reduces specific radioactivity. In an alternative method, which gave higher specific radioactivity, [11C]methane undergoes a free radical iodination in a circulating gas phase while the [11C]methyl iodide formed is continuously trapped on a solid phase to prevent further iodination (Larsen et al., 1997).

11CO2

LAH

» 11 CH 3 OH

H

' »

11

CH3I -«-!?—11CH4 «N'/h2 "COg

Scheme 1. Syntheses of [ 11 C]methyl iodide The reactivity of the electrophilic carbon can be further modulated by preparing [11C]methyl triflate from methyl iodide by an on-line process (Jewett, 1992). Methyl triflate is more reactive than methyl iodide and usually gives higher radiochemical yield (N&gren et al., 1995). Furthermore, the use of [11C]methyl triflate increases the selection of solvents suitable for alkylation reactions. An alternative method for [11C]methyl

ASPECTS ON THE SYNTHESIS OF 11 'C-LABELLED COMPOUNDS

147

inflate synthesis is a gas phase reaction of [11C]methyl bromide prepared from [11C]methane (Mock et al,f 1999). Conversion of the eletrophilic methyl iodide to the nucleophiles methyl lithium (Reiffers et al, 1980) and lithium [11C]methyl(2-thienyl)cuprates (Kihlberg et al., 1997, Neu et al., 1995) broadens the spectrum of functionalities which may be labelled by a methylation reaction. Further transformation of [11C]methyllithium to trimethylsilyl chloride is another method for methylation (Roeda et al., 1999). Diazomethane is another useful but not easily accessible methylating agent (Crouzel et al., 1987b). [ 11 C]Methylhypofluorite, prepared from ["Cjmethyl iodide, was used for the formation of enolmethyl ethers (McCarthy et al., 1993). Other interesting labelling agents prepared from methyl iodide are triphenylarsonium [ ll C]methylide, used in ringclosure reactions (Zessin et al., 1999) and [11C]methylmagnesium iodide (Elsinga et al., 1995) for Grignard reactions. Some of the useful precursors irs prepared from [ ll C]methyl io iodide, are shown in Figure 3, 11

ll

11

CH3OF

l~ 1 CH 3 CuLJX

~*

11

CH3Li

CH31

S 11

CH3N02 "

11

CH3Tf

Figure 3. Examples of precursors obtained from [11C]methyl iodide Tf= triflate Higher alkyl halides are prepared by reacting [11C]carbon dioxide with a Grignard reagent followed by reduction with lithium aluminium hydride and iodination with hydroiodic acid. These reagents do, of course, suffer from the typical isotopic dilution problems encountered with the Grignard reagents or lithiumalkyl compounds. 11C-Labelled ethyl-, allyl-, propyl-, cyclopropyl-, isopropy!-, butyl-, isobutyl-, benzyl- and some derivatives of benzyl halides are examples of alkylating agents prepared by this approach (Langstrom et al., 1986; Fasth et al., 1990b; Antoni & Langstrom, 1987a,b,c; Lasne et al., 1991; Luthra et al., 1985). Apart from the benzylic halides, their reactivities towards nucleophiles are considerably lower than methyl iodide. The synthesis of ethyl iodides is especially problematic since the concomitant production of both methyl and isopropyl iodide gives rise to product mixtures. However, [ 11 Cjethyl iodide was prepared with higher specific radioactivity by a palladium catalyzed reaction of [11C]carbon monoxide with methyl iodide followed by reduction and iodination with hydroiodic acid (Eriksson & Langstrom, 2001). Nitro[11C]alkanes were obtained from labelled methyl and ethyl iodide by reaction with silver nitrate in an on-line process (Schoeps et aL., 1989). The functionality in nitroalkanes gives new options for labelling chemistry (Schoeps & Halldin, 1992). Sulphonarnides and methane sulphonates have been prepared from [11C]methane sulphonyl chloride (McCarron et al, 1999). Formaldehyde is a versatile one-carbon precursor and should thus be an attractive tool in labelling chemistry. It has, however, been rather scarcely used possibly due to lack of a reliable and simple production method. The main applications have so far been reductive formulations of amines, a method superseded by methyl iodide alkylations. [''QlFormaldehyde can be obtained by enzymatic or synthetic approaches (Christman et

148

HANDBOOK OF RADIOPHARMACEUTICALS

al., 1971; Hughes & Jay, 1995). The chemical methods rely on either the formation of [11C]methanol from [11C]CO2, followed by oxidation by ferric molybdenum oxide, silver wool or xenon difluoride (Berger et al., 1980b; Marazano et al., 1977; Nader et al., 1997) or by reduction of [11C]carbon dioxide with metal hydrides at temperatures below -50°C (Nader et al., 1998; Roeda & Crouzel, 2001). In the latter method the low temperature minimized the reduction of formaldehyde to methanol. Formaldehyde has also been prepared in situ and employed in ring-closure reactions (Nader & Oberdorfer, 1999b). Lithium [11C]trimethylsilylynolate may also be used for ring-closure reactions to prepare heterocyclic compounds (Nader & Oberdorfer, 1999c). Aromatic aldehydes have been prepared from the corresponding carboxylic acids by reduction to the alcohol followed by selective oxidation (Halldin & Langstrom 1984a; Kutzman et al., 1980). Acetone is a useful precursor for labelling of AMsopropyl groups as was exemplified in the synthesis of the 11

C-labelled p-adrenergic receptor ligand pindolol (Prenant et al., 1986). [11C] Acetone was prepared by reaction of [11C]CC^ with either methyllithium or methylmagnesium bromide (Berger et al., 1980a). Alkenes are potentially useful precursors for further transformation to other labelling tools as exemplified by the synthesis of l,2-dibromo["C]ethane and [11C]chloroethyltriflate from ethylene obtained via [11C]ethanol by passage through heated quartz glass (Shah et al., 1994a; Shah et al., 1997; Prenant et al., 1995b). Some propenic acid derivatives have also been prepared (Lasne et al., 1992). A classical alkene synthesis, the Wittig reaction, has been used with 11C-labelled methyl iodide (Kihlberg et al., 1990; Grierson et al., 1993; Ogren et al., 1995a) and halonitriles (Homfeldt et al., 1994a, b, c). A difficulty encountered is the problem of adding an equimolar amount of base with respect to the amount of alkyl halide. The problem was overcome by the use of epichlorohydrine, which forms one equivalent of base in situ by the reaction with iodide ions, obtained from the reaction of alkyl iodide with triphenyl phosphine. The carboxylic acid salts obtained in Grignard reactions give the option of acyl chloride preparation, useful for labelling of carbonyl positions. A few examples of these versatile reagents are reported and of these acetyl- (Luthra et al., 1990), cyclohexanecarbonyl-(McCarron et al., 1996), and furoyl chloride (Ehrin E. et al., 1988) are the most elegant examples. [11C]Propyl ketene (Imahori et al., 1989, 1991), prepared by pyrolytic decomposition of ["CJbutyric acid, methylisocyanate (Bonnot-Lours et al., 1993; Crouzel et al., 1995) prepared either from [11C]acetyl chloride or [11C]phosgene, and ["C]methylchloroformate (Ravert et al., 1995) synthesized from [11C]methanol and phosgene are other examples of acylating agents. Urea is also valuable as a labelling reagent particularly with respect to ring-closure reactions. It has been synthesized by several methods starting either with [ !1 C]cyanide (Emran et al., 1985) or [11C]carbon dioxide (Chakraborty et al., 1997). Another versatile precursor is hydrogen [11C]cyanide (Scheme 2), prepared from [11C]methane by catalytic hydrogenation of [11C]carbon dioxide followed by a platinum catalyzed reaction with ammonia (Christman et al., 1975;Iwata et al., 1987). 11CQz

Ni/H21

Pt/NH3 >

Scheme 2. Synthesis of hydrogen [ CJcyanide

ASPECTS ON THE SYNTHESIS OF 11c--LABELLED COMPOUNDS

149

The possibility to transform the nitrile functionality into a carboxylic acid, primary amine or amide introduces multi-functionality through the intermediate nitrile. Hydrogen [HC]cyanide is obtained with a specific radioactivity higher or similar to that of [11C]methyl iodide prepared by the gas phase method. It is further transformed to Cu[11C]CN by reaction with CuSO4/Na2S2O5 and used for aromatic substitution reactions (Ponchant et al, 1997). Ketones (Kothari et al., 1986), halonitriles (Hornfeldt et al, 1992), acrylonitrile

(Antoni & Langstrom

1992),

cyanomethyl pivalate (Thorell

et

al.,

1994) and

11

[ C]rifluoroacetomtrile (Roeda et al., 1999a) are examples of other derivatives prepared from hydrogen [11C]cyanide which are of potential interest as precursors in labelling synthesis. Some of the potentially useful labelling reagents prepared from hydrogen [11C]cyanide are shown in Figure 4. J1 ROO"CCOOR

Ar11CN ->- Na11SCN 11

CM"

11

O li

H2N CNH2 .11, BrCH2(CH2)n"CN 8

RCH=CH11CN KO11CN 11

CNBr

Figure 4. Possible synthetic transformations from hydrogen [nC]cyanide In cyanogen bromide the carbon has reversed polarity as compared with cyanide and is accordingly used as an electrophilic equivalent of the nucleophilic cyanide. This so called umpolung offers new alternatives in labelling synthesis for the introduction of a cyano group. [11C]Cyanogen bromide is prepared by reaction of hydrogen [11C]cyanide with bromine (Westerberg & LSngstrom, 1993, 1997b) or by an on-line process (Westerberg & Langstrdm, 1997a) using perbromide. [11C]Cyanogen bromide has been used as a precursor for the unspecific labelling of macromolecules and for position specific labelling of cyanamides, cyanates and thiocyanates by reactions with amines, alcohols and thiols, respectively (Westerberg & Langstrom, 1994a). Some examples of possible synthetic transformations from [11C]cyanogen bromide are shown in Figure 5.

150

HANDBOOK OF RADIOPHARMACEUTICALS

,11 R-O"CONH 2

11

R-O CN R 1 1 CN NH

R-NH

11 CNBr

RNH-

11

11

CNH 2

NH 11 II R-NH"CNHR'

CN

R-NH 1 1 CONH 2

11

R-S 1 1 CN

S CNT R-SCONH2 Figure 5. Possible synthetic transformations from [11C]cyanogen bromide The carbonyl functionality is a prevalent structure in many biomolecules. The option to have a general method for labelling in this position should give access to many compounds that otherwise might be difficult to prepare. [11C]Phosgene (Scheme 3) was used for this purpose but the employment of this reactive species is restricted to compounds where the two structural units to be coupled to the carbonyl are identical or of very different reactivity (Roedaetal., 1978, 1981). The specific radioactivity of [11C]phosgene has been relatively low, probably due to carrier found in the chlorine gas. Ni/H

O < ii

cr'cci

."CO

-*5L11C02

Scheme 3. Methods for the synthesis of [11C]phosgene [11C]Carbon monoxide was until recently rarely used as a precursor due to difficulties encountered with its low reactivity and low solubility in most solvents. It is prepared by reduction of [11C]CO2 using Zn (Christman et al, 1975), charcoal (Al-Qahtani & Pike, 2000) or molybdenum (Zeisler et al., 1997). The use of solid phase supports for the concentration of [11C]CO2 and [11C]CO and a micro-autoclave for the transition metal mediated carbonylation reactions has solved some of the earlier problems that restricted the use of this easily prepared precursor (Kihlberg & Langstrom, 1999; Kihlberg et al., 200la).

ASPECTS ON THE SYNTHESIS OF 11 C-LABELLED COMPOUNDS

151

ENZYME IN LABELLING SYNTHESIS The use of enzymes as catalysts in labelling synthesis has proven to be an efficient way of synthesizing compounds that would otherwise be difficult to label in the desired position and with sufficient radioactivity to be useful as tracers with PET. Regio-, chemo-, stereo- and substrate selectivity are the typical features usually exhibited by enzymes. Another important consideration, when sensitive substrates are used, is that the syntheses can be performed in aqueous solutions and the conditions employed are close to physiological with respect to temperature and pH. Enzymes are either used free in solution or immobilized on a solid support. Advantages of the latter are a possibility for repeated use and the facilitated separation of the product from the enzymes. Two different types of enzymatic transformations have been distinguished, i.e., carbon-carbon bond formations and functional group transfer or transformation. The latter can be exemplified by transaminations or oxidations of amino groups to ketones (Barrio et al., 1982a; Halldin & Langstrom, 1986). A few generally useful precursors, such as pyruvate and acetyl coenzyme A (acetyl CoA), are available by enzymatic catalysis. The functionalized three-carbon intermediate pyruvate was prepared by several enzyme combinations from racemic alanine labelled either in the carboxylic (Hwang et al., 1986; Takahashi et al., 1990) or 3-position (Bjurling et al., 1988; Ropchan & Barrio, 1984; Cohen et al., 1980). Pyruvate is a versatile compound for further transformations to aromatic amino acids and to lactic acid (Kloster & Laufer, 1980; Bjurling & Langstrom, 1989; Cohen et al., 1980). It is also available by ordinary chemical methods (Kilbourn & Welch, 1982; Tewson et al., 1989). Both aliphatic and aromatic amino acids have been labelled with 11C employing enzymatic and combined chemo-enzymatic methods. The excitatory amino acids L-aspartate (Barrio et al., 1982b) and L-glutamate, the latter labelled in either of the two carboxylic groups, (Cohen et al., 1982) are examples of multienzymatic syntheses of amino acids, starting from [11C]carbon dioxide. Another important development in the field of enzyme-catalyzed reaction was the multi-enzymatic synthesis (Scheme 4) of the aromatic amino acids, 3,4-dihydroxy-L-phenylalanine (DOPA), L-tyrosine, L-tryptophan, 5-hydroxy-L-tryptophan (5-HTP) and analogues thereof, 11C-labelled either in the carboxylic- or B-position (Bjurling & Langstrom 1989; Bjurling et al., 1989, 1990a, b). These chemo-enzymatic syntheses were performed either by free enzymes in solution or by immobilized enzymes (Sasaki et al., 1999; Harada et al., 1999; Ikemoto et al., 1999). DOPA and 5-HTP are of special interest since they are the endogenous precursors for the neurotransmitters dopamine and serotonin respectively. The option of a method that gives access to DOPA and 5-HTP labelled in either of two positions is of pivotal importance for the elucidation of the biosynthesis of dopamine and serotonin. The opportunity to visualize an individual enzymatic step in vivo is given by the fact that the two labelling positions give different labelled products after metabolism and hence radioactive distribution in vivo (Tedroff et al., 1992). = denotes the 11C position

HANDBOOK OF RADIOPHARMACEUTICALS

152 H11CN

* COOH CH2CH

NH, l-tyrosinase COOH

I H

3

DAO/GPT

CCH

.

Q

^j Q

.

H3 CC COOH

NH2 tryptophanase

CH3I

'R-H, HO

Scheme 4. Chemo-enzymatic synthesis of aromatic L-amino acids

C-labelled in two positions

[11C]Phenylalanine has been prepared by the use of a biosynthetic approach (Labarre et al., 1991). Other examples of chemo-enzymatic syntheses that give access to several labelled amino acids are presented in Schemes 5a and b. Enzyme catalyzed nucleophilic substitutions with [11C]cyanide are followed by transformation of the intermediary nitriles to L-2,4-diamino[4-11C]butyric acid, L-[4-11C]aspartate and L-[511 C]glutamate (Antoni et al., 1997, 2001). The same enzyme used to prepare 4-cyano-2-aminobutyric acid was also used in the synthesis of L-[methyl-11C]methionine as well as the ethyl and propyl analogues, ethionine and propionine (Kaneko et al., 1999). Labelled methyl-, ethyl- and propyl sulfides prepared from the corresponding [11C]alkyl iodides were utilized in these amino acids. O

COOH

11

H2N C(CH2)nCH NH2

11 CN"

COOH I +

HCNH2

(CH2)n

Oaeetyl-L-serine sulfhydrylase Oacetyl-L-homoserine sulfhydrylase

COOH 11 I N11C(CH2)nCH NH2

NH2

OCCH3

O COOH HOO11C(CH2)nCH NH2

n=1,2

Scheme 5a. Chemo-enzymatic syntheses of aliphatic amino acids

COOH H2N11CH2(CH2)nCH

ASPECTS ON THE SYNTHESIS OF 11C-LABELLED COMPOUNDS

153 COOH R11CH2SCH2CH2CH NH 2

R= H, C H 3 , C H 3 C H 2

Scheme 5b. Chemo-enzymatic syntheses of aliphatic amino acids L-[3-11C]Serine was synthesized by a reaction sequence including the enzymatic transformation of [11C]methanol to [11C]formaldehyde followed by the formation of L-[3-11C]serine by serine-O-methyl transferase (Svard et al., 1990). Acetyl coenzyme A ([11C]acetyl-CoA) (Mannens et al., 1988) can be used as a general acetylating agent as was exemplified in the synthesis of [11C]acetyl-L-serotonin (Mannens et al., 1990a) and acetyl-L-carnitine. Acyl-carnitines are either labelled in the methyl or carbonyl position in the acyl group or in a methyl group of the carnitine moiety (Angelini et al., 1999; Holschbach et al., 199la). Chemo-enzymatic syntheses have been developed where acetate labelled either in the carboxylic (Pike et al., 1982a) or 2-position (Kihlberg et al., 1994a) and carboxy-labelled propionate was enzymatically converted to the corresponding acyl-L-carnitines (Spolter et al., 1979; Davenport et al., 1997; Jacobson et al., 1997). Enzymatic steps in the synthesis of carbohydrates have also been employed as was demonstrated in the synthesis of 1-11C-labelled D-fructose and D-glucose from mannitol and glucitol, respectively (Ogren and Langstrom 1998) and /V-acetyl-D-[11C]glucosamine via "C-labelled acetyl CoA (Mannens et al.,1990b). Another example is the synthesis of [11C]epinephrine, which was obtained by an enzyme catalyzed rnethylation using L-[11C]methionine as the methyl donor (Soussain et al., 1984). The preparation of Sadenosyl-L-[11C]methionine (Guegen et al., 1982) is another achievement using enzyme catalysis. The same rnethylation reagent was also used for the synthesis of [11C]daunorubicin (Eriks-Fluks et al., 1998). The nucleosides [11C]thymidine and [11C]-2'-arabino-2'-fluoro-a-5-methyl-uridine have been prepared by enzymes immobilized on hollow fiber membranes using [11C]formaldehyde as the labelled precursor (Hughes et al, 1995; Hughes & Jay, 1995). TRANSITION METAL MEDIATED REACTIONS General The most important reactions in organic chemistry, C-C bond formations, are facilitated by transition metals. Organo-transition metal reagents are much weaker bases and nucleophiles than other organo metallic reagents, and easily coordinate a variety of different ligands. They are also tolerant to most functional groups. In 11C-Iabelling synthesis the catalytic aspect is of less importance, since all reagents including the transition metals are used in large excess compared to the labelled precursor. For this reason, the term "mediated" is more appropriate than "catalysed". Palladium is the most successfully applied catalyst among the transition metals. One limitation is encountered with organohalides having B-hydrogens bound to sp3-carbons. These usually give low yields due to the

154

HANDBOOK OF RADIOPHARMACEUTICALS

competing P-hydride elimination reaction (Collman et al., 1987; Yamamoto, 1986; Stille 1986). Organocuprates are much less prone to P-hydride elimination reactions and are useful in cross-coupling reactions with alkyl halides. A disadvantage is that they are strongly basic and functional groups having acidic protons need protection. Cyanations The introduction of a "C-cyano group into an aromatic ring can be performed using palladium as in the synthesis of an analogue of the NMDA receptor antagonist MK-801 (3-cyano-MK801) by cyanation of 3iodo-MK-801 (Andersson et al., 1998). The synthesis of labelled benzamide compounds, as potential tracers for poly (ADP-ribose) synthetase, by Pd catalysed cyanations followed by selective hydrolysis to the corresponding amides is another example of the utility of this reaction (Andersson et al., 1994). Aromatic cyanations with tricarbonylchromium attached to the ring have also been used to synthesize some labelled aromatic nitriles in high radiochemical yield (Balatoni et al., 1989; Andersson & Langstrom, 1994) Cross-couplings Palladium(0)-complexes can catalyze the cross-coupling of an organic halide or triflate with an organometallic reagent in a highly regio-and stereoselective way. These versatile reactions are performed with organostannanes or organoboranes as nucleophiles and are commonly referred to as Stille and Suzuki couplings respectively (Suzuki, 1991). Ketones are obtained if carbon monoxide is present in the Stille reaction. This gives the possibility to label an aromatic methyl ketone in different positions by using either [11C]carbon monoxide or [11C]methyl iodide as the labelled reagent, as shown in Scheme 6 (Andersson et al., 1995; Andersson & Langstrom, 1995 a, b,).

1. CO 2. PhSn(CH3)3

CH3I + Pd(L4) PhSn(CH3)3

* = indicate labelling position with

C.

Scheme 6: Stille couplings The Stille coupling (Suzuki et al., 2000) has been used for the preparation of the isocarbacyclin methyl ester derivative, an antagonist for the CNS type of prostacyclin receptors, as shown in Scheme 7 (Bjorkman et al., 1998, 2000a). Since prostacyclines and prostaglandines do not cross the blood-brain barrier (BBB) the corresponding methyl ester was prepared as a pro-drug.

ASPECTS ON THE SYNTHESIS OF

11

C-LABELLED COMPOUNDS

OCH3

OCH3

SnBu3

Pd(0),

11,

11

CH3I

Scheme 7: synthesis of an isocarbacyclin methyl ester derivative The concept of using methyl esters of 11C-labelled prostaglandins to increase the BBB penetration was evaluated with the label in the methyl ester position (Gullberg et al., 1987). Having the label on the tin moiety, i.e., the labelling reagent, would avoid the problem of having to prepare tin derivatives of large and sensitive biomolecule precursors. 11C-Labelled l-aza-5-stannabicyclo[3.3.3] undecane (Scheme 8) has been prepared as a general precursor in Stille couplings (Vedejs et al., 1992; Forngren, 2001).

11

CH3Li -72°C

Phi, P d ( 0 ), _____

CH

Scheme 8: Synthesis of 11C-labelled l-aza-5-stannabicyclo[3.3.3] undecane The Heck reaction, i.e., the palladium-promoted coupling of organoelectrophiles with alkenes, is another mild and versatile method for C-C bond formations. Its value for 11C-labelling has been investigated using 11 C-labelled alkenes obtained via a Wittig reaction from [11C]methyl iodide (Bjorkman & Langstrom, 2000). This approach permitted the incorporation of 11C into the backbone of carbon structures inaccessible by other methods. Carbonylations The inherent properties of carbon monoxide (i.e., low reactivity and low solubility) have precluded the use of [11C]carbon monoxide as a labelling agent. Some examples of its use have been reported, such as carbonylations of lithium dialkylamides (Kilbourn et al., 1983) and insertion of [11C]CO into a carbon-boron

156

HANDBOOK OF RADIOPHARMACEUTICALS

bond (Tang et al., 1979). Recent technical innovations, where the [11C]carbon monoxide is either trapped on a solid support and the following reaction performed in a micro-autoclave or recycled through the reaction mixture, have greatly diminished the trapping problem (Lidstrom et al., 1997a; Kihlberg & Langstrom, 1999). Compounds where the carbonyl group is bound to carbon have been synthesized using palladium and compounds where the carbonyl group is bound to two heteroatoms were synthesized using selenium. The palladium-mediated carbonylations proceed via a palladium acyl complex, which react with nucleophiles to form a wide range of carbonyl compounds. Some chemical transformations that has been performed with [11C]carbon monoxide using palladium or selenium as catalysts are shown in Scheme 9 (Kihlberg & Langstrom, 1999; Kihlberg et al., 2001a; Kihlberg et al., 2001b; Nader & Oberdorfer, 1999a). R'NH:

RNH 11 CONHR'

R'OH

RNH11CONR",R"'-«—R"'R'"NH—^

R,R'N

11

R

R

CONR"R'"« "- '"

NH

/

Se

-

QF

RY 1 1 COSe'

^

RYH

^^

COOR'

^RNH 1 1 COOR'

R,R'N 11 COOR"

11CO HNR'R"

R11COR'

RPd(PPh 3 ) 2 X

*- R^CONR'R'

R 1 1 COPd(PPh 3 ) 2 X

Scheme 9: Possible chemical transformation using [ 11 C] Carbon monoxide In most cases, aryl halides have been used although aryl triflates may also be employed. The latter usually give lower radiochemical yields but can be used if lithium bromide is present. Diaryliodonium salts (e.g., diphenyliodionium bromide) have, however, been shown to be an interesting alternative in the synthesis of 11

C-labelled ketones (Al-Qahtani & Pike, 2000).

Amides are readily labelled using palladium mediated carbonylations (Kihlberg & Langstrom, 1999). The corresponding labelling of open esters gives significantly lower yields due to the weaker nucleophilicity of alcohols compared to amines (Kihlberg et al., 2001a). Aldehydes are efficiently labelled at relatively low temperatures using different hydride donors as the source of nucleophile (Bjorkman, et al., 2000b). Labelled aldehydes and ketones provide another route to obtain functionalized alkenes via the Heck reaction (Bjorkman & Langstrom, 2000). Flavones and phtalimides can be synthesized by 11C-carbonylations in ring-

ASPECTS ON THE SYNTHESIS OF 11C-LABELLED COMPOUNDS

157

closure reactions (Kihlberg et al., 1999; Karimi et at., 2001). 11C-Labelled carbamoyl compounds can be prepared by using a selenium-mediated reaction with [11C]carbon monoxide. The urea formation only works satisfactorily with primary amines or in case of cyclisations (Kihlberg et al.,2001a, 2001b). Since selenium is practically insoluble in most solvents the use of primary alkyl amines or tetrabutylammonium fluoride is necessary for the formation of soluble and reactive complexes with selenium. Copper mediated reactions In order to label carboxylic acids in positions other than the carboxylic acid group, methods using cuprate mediated coupling reactions were developed. Bis-Grignard reagents and di-lithium tetrachlorocuprate were used in coupling reactions with 11C-labelled alkyl iodides in the syntheses of a broad range of saturated fatty acids and arachidonic acid (Scheme 10) (Kihlberg & Langstrom, 1994a, b; Kihlberg et al., 1994b). An alternative to this is the use of in situ-prepared organo cuprates, obtained from eo-iodo fatty acid esters. (Neu et al., 1997a; Wust et al., 2000). R11CH21

+ XMgR'MgX

R'--(CH2)p-,

Cu(l) - LL*-

11 t.COo 11 R 1 1 C H 2 R'M gX - -~+~ R 1 1 C H 2 R'C O 2 H

-(CH 2 ) 3

R = H-, C H 3 - , C h 3 C H 2 - , C H 3 ( C H 2 ) 7 -

P =3, 4, 5, 6, 6, 10, 12, 14, 16

Scheme 10: Syntheses of saturated and ansaturated fatty acids Another important application for these nucleophilic alkyl reagents is exemplified by the precursors, lithium [11C]methyl(2-thienyl)cuprates (Kihlberg et al., 1997), and their use in the synthesis of [21-11C]progesterone and [la-methyl-11C]mesterolone (Scheme 11) (Lidstrom et al., 1997b; Neu et al., 1998).

L i 1 1 C H 3 ( 2 - T h ) C u (LiCN) TMSCI

O

OH

OAc 11

CH3

. L i H C H 3 ( 2 - T h ) C u (LiCN)

O

1

1

J

1

Scheme 11: Synthesis of [21- 11 C] progesterone and [la-methyl-11C] mesterolone

HANDBOOK OF RADIOPHARMACEUTICALS

158

RING-CLOSURES Labelling a carbon in an aromatic ring may give access to useful structural units with the label in a metabolically stable position. The synthesis of some aromatic compounds including heterocycles are shown in Scheme 12 (Zessin et al., 1999; Mading et al., 1997, 2000; Mading & Steinbach, 1998, 2000; Steinbach et

al., 1995 ).

M e 2 N H 2+

cr

11

R 1 = E t, C H 2 O H R 2 = t-B u, P h

C H3N O2

NH

NO2

*= denotes the position of 11C Scheme 12: Labelling of aromatic compounds in the ring structure A multistep synthesis of the putative glycine antagonist Licostinel based on the [11C]quinoxaline ring structure, from diethyl[11C]oxalate is shown in Scheme 13 (Thorell et al., 1993b, 1995, 1998). Although the radiochemical yield was rather low, the method is interesting since it offers a general method for

11

C-

labelling of the quinoxaline structure.

CH3OCOCI

+

11 ''CN

CH3CH2OCO

11 11

COOCH2CH3

NO2

Scheme 13: Synthesis of Licostinel Nucleosides have been prepared by different ring-closure reactions as is exemplified by the synthesis of 1,3,6-trimethyl [2-11C]uracil from 1,3-dimethyl [2-11C]urea (Castro-Iberra et al., 1997).

ASPECTS ON THE SYNTHESIS OF 11 C-LABELLED COMPOUNDS

159

A procedure for 11C-labelling of the 4-imidazolyl group using [11C]formaldehyde is shown in Scheme 14. The 4-imidazolyl group is commonly found in histamine receptor ligands (Roeda et al., 1997).

0

0 H

H

H

2.H2S,HOAc

O

H

Scheme 14: Synthesis of 4-imidazolyl derivatives An acid catalysed ring-closure of 11C-labelled cyanopropanol to the corresponding butyrolactone busulphan has also been described (Hassan et al., 1991). THE SYNTHESIS OF RADIOTRACERS FOR PET SYNTHESIS OF COMPOUNDS FOR THE STUDY OF RECEPTORS AND ENZYMES General considerations Normal brain functions are maintained by a complex interplay of around 100 neurotransmitters acting on an even larger number of receptor subtypes. A multitude of receptor ligands have been prepared and used as PET tracers, however a detailed presentation of these is outside the scope of this chapter. The possibility to probe the existence and to be able to measure the concentration of a certain enzyme in vivo is another important PET application. This is achieved through the use of labelled enzyme substrates or inhibitors. From a chemical point of view the chemistry used to prepare most of the receptor ligands is similar, with [11C]methyl iodide alkylations of heteroatoms as the most prominent example. In this chemistry oriented review the main objective is to give some examples of the compounds available and indicate some methodologically important synthetic achievements. Dopaminergic ligands Most attention has been focused on ligands for the dopamine-D1 and D2 receptors (Volkow et al., 1996). The first example of a dopamine-D2 antagonist that could visualize striatal dopamine receptors was N-[methyl11 C]methylspiperone (Wagner et al., 1983). This compound also has affinity for the 5-HTA2 receptors, but has now been superseded by the more dopamine-D2 selective receptor ligand [11C)raclopride (Scheme 15) (Ehrin er al., 1986).

HANDBOOK OF RADIOPHARMACEUTICALS

160

o OH

H311CQ base/ CH3I OH

Scheme 15: Synthesis of [11C]raclopride The presynaptic dopamine receptor antagonist (-)-OSU6162 was labelled with 1!C and used to investigate pharmacologic effects of the dopminergic system (Neu et al., 1997b). Only a few labelled antagonists are available for the study of dopamine-D1 receptors, these include SCH23390 (Halldin et al., 1986), NNC220215 (Foged et al., 1998) and NNC112 (Halldin et al., 1998). Receptor agonists that can be used for the in vivo assessment of the high affinity state of the receptor are also of interest. Propyl- (Hwang et al., 2000) and methylnorapomorphine (Ziljstra et al., 1993), pramiprexole (Hwang. et al., 1999) as well as some tetralin derivatives have been prepared as potentially useful D2 receptor agonists (Shi et al., 1999). Some agonists for the D1 receptor are also available (DaSilva et al., 1999). The extrastriatal dopamine receptors need a tracer with higher affinity than the ligands mentioned, and the antagonist FLB457 was prepared for the study of these (Halldin et al., 1995). There have been several compounds developed for the study of the dopamine transporter, these include cocaine (Fowler et al., 1989), nomifensine (Ulin et al., 1989; Aquilonius et al., 1987), B-CIT (Farde et al., 1994), B-CIT-FE, P-CIT-FP (Halldin et al., 1996; Lundkvist et al., 1995), and WIN35,428 (Wong et al., 1993). Serotonergic ligands The serotonin transporter is a target for most of the recently developed antidepressant drugs. The therapeutically effective drug citalopram was 11C-labelled and used for in vivo imaging but without any promising results (Dannals et al., 1990). [11C]McN5652 may, however, be more suitable for the study of the serotonin transporter (Suchiro et al., 1993; Huang et al., 1998). The HTIA antagonist [11C]WAY100635, used for imaging of the 5-HTIA receptors in the brain, is a good example where the labelling position is of importance. Labelling in the methoxy position (Mathis et al., 1994) resulted in the formation of the lipophilic labelled metabolite WAY 100634 that also penetrated the BBB and added to the non-specific binding in the brain (Osman et al., 1996). This problem was solved by the development of a synthetic route for labelling in the carbonyl position, which is shown in Scheme 16 (McCarron et al., 1996).

ASPECTS ON THE SYNTHESIS OF 11 C-LABELLED COMPOUNDS MgC1

I.HCP,

161 COCI

2. SOCl2

H3 CO CH3I

Scheme 16: Synthesis of WAY 10063 5 labelled in the carbonyl or methoxy positions Some halogenated analogues of WAY100635 have also been 11C-labelled and evaluated as tracers (Sandell et al., 2001). Similar problems with metabolites that disturb the interpretation were encountered using the selective 5-HT2A antagonist [11C]MDL100907 (Lundkvist et al., 1996). The methoxy group in the 3-position was chosen for labelling since the 3-OH analogue (MDL10575) had been identified as a metabolite. Labelling in position 2, should thus give a labelled compound with unknown biological properties. [11C]LU29-066 a 5-HT2 receptor antagonist was prepared from [11C]phosgene (Amokthari et al., 1995). For the study of 5-HT3 receptors some antagonists (Rajagopla et al., 1992) have been prepared such as [11C]MDL72222 (Barre et al., 1992) and [11C]granisetron (Vandersteene et al., 1998) as well as the agonist [11C]LY274601 (Suehiro et al., 1998) and the partial agonist [11C]S21007 (Guillout et al., 1996). GABAergic ligands GAB A itself has been labelled but the poor BBB penetration excludes its use for the study of the GABAergic system (Antoni & Langstrom, 1988). Analogues of GABA such as methoxyprogabic acid (De Vos Siegers 1994) may be an alternative. [11C]Flumazenil (Samson et al., 1985) has proved to be a useful tracer for the study of benzodiazepine receptors. Alternatively the well-known pharmaceutical diazepam can be used (Sassaman et al., 1999). R[11C]PK11195 has been established as a useful tracer for the study of peripheral benzodiazepine receptors (Shah et al., 1994b). Adrenergic ligands The study of the adrenergic system has been an important area of PET research (Syrota. 1991; Syrota & Merlet, 1996). Early attempts to visualize the adreno-receptors utilized 11C-labelled atenolol, metoprolol (Antoni et al., 1989), propanolol (Berger et al., 1982) and (S)-[11C]CGP12177 (Delforge et al., 1991). More successful approaches for the study of adrenergic receptors included the a1 antagonist [11C]GB67 (Law et al., 2000) and the B1 ligand [11C]CGP20712A (Elsinga et al., 1994). As alternatives 11C-Labelled metaraminol (Nagren et al., 1994) and m-hydroxy[11C]ephedrine (Rosenspire et al., 1990), sympathomimetic compounds

162

HANDBOOK OF RADIOPHARMACEUTICALS

acting as false neurotransmitters, have been used. [11C]Phenylephrine (De Rosario & Wieland, 1995) and miodobenzylguanidine are other compounds useful as functional heart neuronal markers. The latter is prepared from cyanogen[11C]bromide which gives the label in the guanidine group (Westerberg & Langstrom. 1997b). Cholinergic ligands [11C]Nicotine (Berger et al., 1979) has been used for the study of nicotinic cholinergic receptors. The flow dependence and a large fraction of unspecific binding of nicotine diminish its potential as a tracer and alternative ligands are being investigated (Kassiou et al., 1997). Other attempts to find a useful nicotinic receptor ligand based on agonists, have also been presented, for example, ABT-418 (Dolle et al., 1996; Sihver et al., 1999). The nicotinic receptor antagonist, 3-quiniclidinylbenzilate, was labelled by an esterification of [11C]benzilic acid obtained from carboxylation of lithiated benzophenonedianion (Prenant et al., 1989). The naturally occurring alkaloid epibatidine, a potent nicotine receptor antagonist, has been used as lead compound for several analogues which were investigated as tracer candidates in PET studies (Patt et al., 1999, 2000; Spang et al., 1999, 2000). Several ligands for the muscarinic acetylcholine receptor have been synthesized. These include N-methyl-4piperidyl benzilate and the corresponding ethyl- and propyl analogues (Mulholland et al., 1988; Nishiyama et al., 2000), N-methyl-3-piperidyl benzilate (Takahashi et al., 1999), (+)-2-tropanylbenzilate (Mulholland et al., 1992) and MQNB (Delforge et al., 1990). A few labelled agonists have been prepared for the study of the muscarinic-M1 receptor namely xanomeline (Farde et al., 1996) and [11C]milameline (Hartvig et al., 1997). The vesicular uptake system is another important function that can be studied with PET. Methylaminobenzovesamicol was labelled with [11C]methyl iodide for this purpose using an activating group on the nitrogen to direct the methylation to the desired position (Mulholland & Jung, 1992). Miscellanous types of receptor ligands Much effort has been invested in the search for selective ligands for the different subclasses of opioid receptors. The u-selective carfentanil and the non-selective diprenorphine are perhaps the most used 11Clabelled opiate receptor ligands (Dannals et al., 1985). Diprenorphine can either be labelled in the cyclopropyl - (Luthra et al., 1985) or the 6-methoxy group (Luthra et al., 1994), the latter being the most convenient labelling position. N-1'-([11C]methyl)naltrindole has been developed as a selective 6 opiate receptor ligand (Madar et al., 1996). For studies of the a opioid receptor [11C]SA4503 (Kawamamura et al., 2000), 1,3-di(tolyl)-[11C] guanidine (Westerberg et al., 1994) and a benzomorphane derivative have been synthesized (Musachio et al., 1994). The k-receptor may be studied by the use of [11C]GR89696 prepared from [11C]methylchloroformate (Ravert et al., 1999). Receptor ligands selective for the different sites of the N-methyl-D-aspartate (NMDA) receptor complex have come into focus in the search for a tool for the study of neurodegeneration caused by ischemic processes and other brain injuries (e.g., stroke). The glycine site specific NMDA receptor ligand [11C]L703,717 is one example where the prodrug concept has been applied (Haradahira & Suzuki,. 1999). Since [11C]L-703,717 poorly penetrates the BBB the corresponding acetyl ester analogue was prepared to improve the brain uptake (Haradahira et al., 1999). Non-competitive NMDA antagonists such as R- and S-

ASPECTS ON THE SYNTHESIS OF 11 C-LABELLED COMPOUNDS

163

[11C]ketamine, have been evaluated as tracers both in rhesus monkey and humans (Hartvig et al., 1994). LY20157, 11C-labelled in the tetrazole group, is another example of a tracer for the NMDA receptor complex (Ponchant et al., 2000). Some xanthine-based ligands such as KF15372, KF17837, KF21213, CSC and KF18446 have been labelled with 11C for imaging the adenosine A1 (Ishiwata et al., 1995a) and A2 (Ishiwata et al., 1996; Ishiwata et al 2000; Marian et al., 1999; Wang et al., 2000) receptors. The neurokinin-land histamine H-1 (Ravert et al., 1993) receptors are also targets for drag development and useful PET tracers for these receptors are available. For the former receptor has the receptor antagonist [11C)GR205171 proved to be a useful PET ligand (Bergstrom et al., 2000). Calcium channel antagonists such as [11C]semotiadil (Ishiwata et al., 1994), [11C]nisoldipine (Holsbach et al., 1991b) and [11C]isrodipine (Crouzel & Syrota 1990) have been 11C-labelled. Tracers for the study of angiotensine-II (Mathews et al., 1995) and endothelin receptors (Ravert et al., 2000) are also available. Tricyclic antidepressants are important pharmaceuticals for regulation of the synaptic concentration of norepinephrine. [11C]Desimipramine and its metabolite 2-hydroxy[11C]desimipramine have been synthesized for the study of the membrane bound norepinephrine transporter (van Dort et al., 1997). Enzyme substrates and inhibitors The brain monoamine oxidase type B enzyme (MAO-B) has been studied using the irreversible inhibitor Ldeprenyl (MacGregor et al., 1987; Fowler et al., 1987) and the enzyme substrate N,Ndimethylphenethylamine (Halldin et al., 1989). The latter was labelled either in the methyl or phenethyl group. The double-isotope labelling approach was employed also for the determination of MAO-B concentration in the heart and the brain using [11C](-)-a,a-dideutero-phenylephrine (De Rosario et al., 1996, De Rosario & Wieland, 1995) and a,a-dideutero-L-[11C]deprenyl, respectively (Fowler et al., 1995). The enzyme inhibitors clorgyline, harmaline and harmine (Westerberg et al., 1995b) are examples of compounds used for mapping the MAO-A enzyme distribution. Of these enzyme inhibitors harmine is the most versatile tool for the study of the brain distribution of MAO-A (Bergstrom et al., 1997). Physostigmine, a potent inhibitor of acetylcholine esterase, was labelled by the use of [11C]phosgene (Crouzel et al. 1995). Other examples of 11C-labelled acetylcholine esterase inhibitors are methyltetrahydroaminoacridine (Tavitian et al., 1993), N-methylpiperidine-4-yl acetate (Iyo et al., 1997) and propionate (Kilbourn et al., 1998; Snyder et al., l999b; Koeppe et al., 1999). The influenza virus neuramidinase inhibitor GG167 labelled with 11C in the guanidine group was synthesized from [11C]cyanogen bromide (Westerberg et al., 1996). The enzyme 11-B-hydroxylase is preferentially found in the adrenal cortex and can be visualized by the selective inhibitor etomidate and its methyl analogue metomidate, prepared from 11C-labelled ethyl- or methyl iodide, respectively (Bergstrom et al., 1999). Tracers have also been developed for mapping the biodistribution of endopeptidases and poly (ADP-ribose) synthase as exemplified by the synthesis of [11C]Y-29794 (Charalambous et al., 1994) for the former and some 11C-labelled benzamides (Andersson et al., 1994) and an isoquinolone derivative (Miyake et al., 2000) for the latter. S-[11C]Methyl-L-thiocitrulline (Zhang et al., 1997), S-[11C]methylisothiourea (Zhang et al.,

164

HANDBOOK OF RADIOPHARMACEUTICALS

1996) and N(W)-nitro-L-arginine[11C]methyl ester (Roeda et al., 1996) have been prepared for the in vivo measurement of nitric oxide synthase. [11C]Diazomethane was used for the esterification in the latter synthesis. Some modified "C-rotenoids have been prepared with the objective to measure mitochondrial complex I activity (Snyder et al., 1999a; Charalambous et al., 1995) for the study of neurodegenerative disorders associated with cellular oxidative metabolism. Tyrosine kinase inhibitors may prove to be useful tracers for the study of tumors with one example being the synthesis of ["C]PD153035 (Johnstrom et al., 1998). Another potent enzyme inhibitor [HC]Vorozole was synthesized for the study of aromatase activity (Lidstrom et al., 1998a). The selective cAMP-specific phosphodiesterase-4 inhibitors, Ro20-1724 and R- and S-rolipram have been 11C-labelled and evaluated in rats as tracers for the signal transduction in the second messenger system (DaSilva et al., 2001).

SYNTHESIS OF 11C-LABELLED ENDOGENOUS COMPOUNDS Amino acid synthesis Amino acids constitute an important class of compounds with a diversity of functions in the body. These include: energy metabolism, building blocks for proteins, neurotransmitters or precursor for neurotransmitters. Disturbances in amino acid metabolism and transport over membranes are related to many diseases. Most of the naturally occurring amino acids have been 11C-labelled using methods that give racemic products. However, the importance of preparing enantiomerically pure amino acids has further encouraged research in the field of amino acid synthesis and some of these methods have been modified for use in 11Clabelling synthesis. The synthetic strategies adopted has been either to establish the stereochemistry in the precursor and introduce the label in a position that does not involve the chiral centre or to separate the enantiomers of a racemic synthesis by chromatographic (Washburn et al., 1982) or enzymatic methods (Barrio et al., 1982a). The enzymatic methods utilize D-amino acid oxidase to oxidise the undesired enantiomer to the corresponding keto acid or the selective removal of an aminoacetyl group liberating the desired unprotected amino acid. The resolution approach inevitably results in a 50% loss of radiochemical yield, and that is of course a serious limitation. The most challenging method is asymmetric synthesis, which also may give the possibility to prepare both enantiomers in pure form. The labelling position in the amino acids is another factor to be considered when designing the synthesis. Different positions may be preferred depending on the intended biological application. An important example of this is the use of L-[B-11C]DOPA and 5-hydroxy-L-[B-11C]tryptophan for the study of neurotransmitter synthesis where decarboxylation is the metabolic step resulting in dopamine and serotonin, respectively. On the other hand, labelling in the carboxylic position is a requirement for the application of 11C-amino acids in the Sokoloff model for brain protein synthesis (Smith et al., 1980).

ASPECTS ON THE SYNTHESIS OF 11C-LABELLED COMPOUNDS

165

The main synthetic strategies that can be adopted for the synthesis of 11C-labelled amino acids are as follows: * Bucherer-Strecker synthesis using either [ 11 C]cyanide or [ 11 C]aldehydes * Carboxylation reactions using [ 11 C] carbon dioxide * Condensation reactions using [ 11 C]aldehydes * Alkylation reactions using [ 11 C]alkyl halides * Enzymatic methods In the high-pressure high-temperature modification of the Bucherer-Strecker

synthesis, hydrogen

11

[ C]cyanide is reacted with the bisulfite adduct of an aldehyde (Barrio et al., 1982b; Hayes et al., 1978; Casey et al., 1981). This synthesis gives carboxy labelled amino acids but is restricted to neutral amino acids. Labelled aromatic aldehydes have also been applied in this reaction (Halldin & Langstrom, 1984b) as well as in condensation reactions with oxazolones (Halldin & Langstrom, 1984c,1986) for labelling in the a- or the p-positions, respectively. L-[3-11C]Phenylalanine was prepared by the use of a chiral hydrogenation catalyst by the latter method (Halldin & Langstrom, 1984d). An alternative method for labelling in the carboxylic position is the direct addition of [11C]CO2 to lithiated carbanions of isonitriles (Vaalburg et al., 1976; Bolster et al., 1986; Hamacher & Hanus, 1989). Alkylation of the stabilised carbanion of protected glycine derivatives with aliphatic or aromatic alkyl halides is a way of labelling in the 3-position (or P in the case of an aromatic amino acids) (Antoni & Langstrom, 1987a, b, c; Kilbourn et al., 1984). The preparation of L[11C]methionine (Langstrom et al., 1987) and L-[4-11C]ornithine (Ding et al., 1989) from [11C]methyl iodide and hydrogen [11C]cyanide, respectively, are examples of syntheses where the chiral centre can be established before introduction of the label. The synthesis of L-[methyl-11C]methionine is based on the alkylation of the corresponding sulphide anion of L-homocysteine. Two of the methods available for generating the sulphide anion of homocysteine for alkylation with [11C]methyl iodide are shown in Scheme 17 (Comar et al., 1976; Schmitz et al., 1995). COOH CH 2 -SCH2CH2CH

NH2

COOH 1.Na/NH3(l)

p.. CH3 , '

1 . NaOH/aceton

9 nrH .

)— NH2

NH2

Scheme 17. Synthesis of L-[methyl-11C]methionine The methionine analogue selenomethionine was 11C-labelled using a similar reaction sequence (Nagren & Langstrom, 1987). Asymmetric syntheses of 11C-amino acids are mainly based on the alkylation of chiral glycine derivative with 11 C-labelled alkyl halides. Synthesis of L-[3-11C]alanine was the first example of an asymmetric amino acid synthesis with 11C (Langstrom & Stridsberg 1979). Since this first attempt other chiral "handles" have been explored and the chiral induction have been improved from the rather modest 48% up to 98% enantiomeric excess (e.e.)(Fasth et al., 1989, 1990a). The method employing [(+)-2-hydroxypinanyl-3-idene]glycine tertbutyl ester as chiral handle was used to prepare some 3-11C-labelled aliphatic amino acids that were obtained with enantiomeric purities in the range of 80-89% e.e. (Antoni & Langstrom, 1986. 1987b). Enantiomeric purities up to 90% e.e. were obtained using a method based on the alkylation of the nickel complex of the

166

HANDBOOK OF RADIOPHARMACEUTICALS

Schiffs base of S-o-[(N-benzylpropyl)amino]benzophenone and glycine (Fasth & Langstrom, 1990). One interesting feature of this method was the possibility to separate the diastereomeric alkylation products and thus prepare nearly enantiomerically pure products. One of the most promising methods, which gives nearly enantiomerically pure 11C-amino acids used an imidazolidinone derivative (Scheme 18) (Fasth et al., 1995; Plenevaux et al., 1994a).

1. Base

\

2. R 11 CH 2 I

/

/

•-—^ r^ N^

Hydrolysis

UPL, Q CH R CH2R

*•

««

R

11

9I O O H

CH2CH NH

BOC

Scheme 18. Asymmetric synthesis of 11C-amino acids by alkylations of imidazolidinone Another possibility is to use a reaction between [11C]cyanide and chiral aziridine derivatives which gives access to 11C-labelled aspartate, asparagines and 2,4-diaminobutyric acid (Gillings et al., 2001). The nucleophilic ring-opening of the aziridines with [ C]cyanide did, however, result in racemic products. Peptides are also interesting as tracers, although so far only a few examples of methods for 11C-labelled peptides have been presented. One general method for peptide labelling requires that an 5benzylhomocysteine residue is present in the protected peptide. Labelling of the peptide is then accomplished by methylation of the corresponding sulphide anion of homocysteine, generated by the use of sodium/liquid ammonia (Franzen et al., 1987, 1988; Langstrom et al., 1981b; Nagren et al., 1986, 1988). Some 11C-labelled amino acid analogues have also been prepared. a-[Methyl-11C]methylphenylalanine and a-[methy-11C]methyltyrosine were synthesized by alkylations of malonic esters (Gee & Langstrom 1991). Some branched aliphatic amino acids have been synthesized, as exemplified by a-aminoisobutyric acid and P-aminoisobutyric acid (Alauddin et al., 1997). a-Aminoisobutyric acid can be labelled in either the carboxylic position (Dunzendorfer et al., 1981) or 2-position (employing [11C]acetone as the labelling reagent) (Prenant et al., 1995a), or the 3-position by a methylation reaction (Oberdorfer et al., 1993; Schmall et al., 1996). Of these amino acid analogues, a-aminoisobutyric acid and a-methyltryptophan have attracted the most attention as potentially useful biological markers (Suchiro et al., 1992; Plenevaux et al., 1994a, b; Mzengeza et al., 1995). Another non-proteinogenic amino acid that has been prepared as a tumor-seeking agent is 1-aminocyclopentane carboxylic acid (Hayes et al., 1976; Iwata et al., 1995). 11C-Labelled paraboronophenylalanine has been synthesized as a tracer for treatment planning in connection with boron neutron capture therapy (Kabalka et al., 2001). Carbohydrates and miscellaneous endogenous compounds Some examples of endogenous amines connected to different regulating systems in the body that have been labelled with 11C include dopamine (Christman et al., 1970), choline (Pascali et al., 2000; Hara et al., 1997), epinephrine (Chakraborty et al., 1993a, b; Nagren et al., 1994), melatonin (Le Bars et al., 1987),

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norepinephrine (Fowler et al., 1974), octopamine (Maeda et al, 1990; Nagren et al., 1994), and tyramine (Schoeps et al., 1993). Carbohydrates such as glucose have been a major interest in the study of in vivo biochemistry. Several methods utilizing [11C]cyanide, [11C]methyl iodide or [11C]nitromethane have been developed for the specific labelling of glucose in different positions, of these the positions 1 and 6 have attracted the most interest (Shiue & Wolf, 1981; Tada. et al., 1989; Schoeps et al., 1991; Grierson et al., 1993; Ogren et al., 1995b, 1997b). Due to the problems encountered with interpretation of the dynamic images from glucose, 2-deoxyglucose was developed as an alternative and the concept of metabolic trapping was developed (Reivich et al., 1982; Padgett et al., 1982a). A method recently presented for the synthesis of 2-deoxy-D-[1-11C]glucose used a Wittig reaction followed by a PdC12 catalysed oxidation (Ogren & Langstrom, 1997a). Another example of a transition metal catalysed reaction is the synthesis of D-[l-11C]glucosamine, where PdCl2 was used to convert the gluconitrile, prepared from [11C]cyanide, to the aminosugar (Thorell et al., 1993a). Fatty acids serve as the major source of energy for the heart as well in the muscle. Efforts to label suitable candidates, mainly in the carboxylic position by [11C]carboxylation of Grignard reagents for heart imaging have produced both saturated and unsaturated fatty acids (Piette et al., 1989; Channing & Simpson, 1993). Of these palmitate (Padgett et al., 1982b) and acetate (Pike et al., 1981; Oberdorfer et al., 1992, 1996; Ishiwata et al., 1995b) have been the most employed and evaluated tracers. B-[11C]Methyl heptadecanoic acid (Livni et al., 1982), 3,3-[11C]dimethylheptadecanoic acid (Jones et al., 1988) and other fatty acids that do not participate in the B-oxidation have been developed to simplify the study of heart metabolism by utilizing the concept of metabolic trapping of the tracers. Labelling in the 2-position of some B-methyl fatty acids was achieved by alkylation of malonic ester with [11C]methyl iodide (Ogawa et al., 1997). In another attempt to reduce the metabolic degradation of the tracer during the course of the PET investigation some phenoxybridged fatty acids were synthesized (de Groot et al., 1997). However, no improvement of the imaging results was obtained. Medium chain fatty acids are carnitine independent for the transport into the mitochondria and some octanoate analogues were developed for the study of deficiency in the B-oxidation of the mitochondria (Kawashima et al., 1997, 1998). A leukotriene derivative, a potent metabolite of arachidonic acid, was 11C-labelled by acetyl chloride for the study of the metabolic pattern of leukotrienes (Oberdorfer et al., 1992). The steroid structure is difficult to build up from simple labelled intermediates within the time frame allowed for 11C synthesis. However, some examples of steroids successfully 11C-labelled include 17-amethyltestosterone (Berger et al., 1981b), 17a-[11C]methylestradiol and 11B-ethyl-17a[11C]methylestradiol (Dence et al., 1994). In these syntheses [11C]methyl iodide was converted to [11C]methyllithiumand was further reacted with the carbonyl functionality to give the corresponding methyl ether. Examples of different labelling strategies are the syntheses of the adrenocortical steroid, triamcininolone acetonide, prepared by reaction with [11C]acetone (Berridge et al., 1994) and [carbonyl-11C]estramustine phosphate (Lidstrom et al., 1998b) synthesized using [11C]phosgene. The copper mediated synthesis of mesterelone and progesterone are presented in Scheme 11. Functional analogues of steroids without the steroid structure have been employed as pharmaceuticals and have also been labelled for use as tracers for PET. The antioestrogenic compounds

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toremifene (Nagren et al., 1991) and tamoxifen have been 11C-labelled by methylation (Svard et al., 1982) for studies on oestrogen-receptor positive tumors. [11C]Tamoxifen was also labelled by a direct incorporation of [11C]carbon dioxide in a reaction with a N-trimethylsilyl derivative (Ram & Spicer, 1989). The same reductive-carboxylation approach was also applied for the synthesis of N-[4-11Cmethyl]imipramine (Ram et al., 1986). The development of tracer for the study of cell proliferation has been an important area of research. The labelling of thymidine in different positions is such an example (Goethals et al., 1992; Alauddin et al., 1995). The methyl position is not optimal since it gives rise to labelled metabolites that interfere with the interpretation of the PET data. Labelling in any of the carbonyl positions may be advantageous since [11C]carbon dioxide is the most prominent labelled metabolite using this labelling position. This has been achieved by using [11C]urea or [11C]phosgene in ring-closure reactions (Labar et al., 1991; Borght et al., 1991; Steel et al., 1993). Other potentially useful tracers for the study of cell proliferation are [11C]uracil (Chakraborty et al., 1997), the uracil analogue [11C]FMAU (Conti et al., 1995), 6-[methyl- 11 C]methyl-2'deoxyuridine (Goethals et al., 1997) and [methyl-11C]p-pseudothymidine (Grierson et al., 1995). MISCELLANEOUS 11C-COMPOUNDS Antibiotics are important Pharmaceuticals and their distribution in vivo are of considerable interest when selecting an appropriate compound for treatment of bacterial infections. Roxithromycine (Barre et al., 1995), a macrolide and the well-known erythromycin-A (Pike et al., 1982b) are examples of 11C-labelling of antibiotics. L- and D-[11C]dimethylcarbamoyl-PAF, platelet-activiting analogues for the study of tumors were labelled by methylation with [11C]methyl iodide (Sasaki et al., 1996). Some 11C-labelled colchicines have been used for drug resistance studies related to overexpression of the P-glycoprotein (Kothari et al., 1995; Levchenko et al., 2000). The sarcosinamide analogue SarCNU (Conway et al., 1988) and some nitrosoureas (Diksic et al., 1985), (cytotoxic agents for chemotherapy), were labelled in the carbonyl position. The antitumor agent DACA has also been 11C-labelled (Brady et al., 1997). Although the majority of 11C-labelling syntheses are position specific some examples of uniform or unspecific labelling are found in the literature. Most of these are unspecific labelling of macromolecules. Methods used for the labelling of proteins include reductive methylation employing [11C]formaldehyde and direct alkylation with methyl iodide (Straatmann & Welch, 1974; Marche et al., 1975; Turton et al., 1984). Labelling with formaldehyde and methyl iodide suffer from the drawbacks that either a reducing agent or alkaline conditions are needed in the synthesis. This may be deleterious to the protein. As an alternative [11C]cyanogens bromide may be used since it can be used at near physiological conditions (i.e., temperature and pH). Proteins such as transferrin and albumin as well as the polysaccharide hyaluronan have been labelled by this method (Westerberg & Langstrom, 1994b; Westerberg et al., 1995a). CONCLUSIONS - FUTURE PERSPECTIVES The advent of positron emission tomography will in a significant way increase our knowledge of the basic processes constituting the chemistry of life. The possibility to perform non-invasive investigations in humans

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to study in vivo biochemistry and eventually follow the fate of a certain atom in a molecule is providing new information that is probably beneficial both in basic science and clinical practice. The PET-camera technology is now close to its theoretical resolution limit whereas the full scopes of the chemistry perspectives still lie in the horizon. Synthesis with carbon one of the most important elements of life, is foreseen to have a nearly unlimited potential for further innovations. This is due to the large library of established synthetic achievements available that can be modified and implemented in 11C-labelling synthesis. The future will probably also give examples of the reverse, i.e., developments in labelling synthesis that are exploring new possibilities in carbon chemistry. The introduction of [11C]methyl iodide nearly thirty years ago as a labelling reagent is one of the milestones of 11C-chemistry. Today we may see the next turning point the exploration of [11C]carbon monoxide as a generally applicable labelling agent. Both these labelled precursors only became really useful in routine work after the technical obstacles in their production were solved and semi-automated synthetic approaches could be employed. The use of transition metal mediated reactions is perhaps the most important new introduction in labelling synthesis and the examples presented in the literature hold promise for continuing progress in this direction. The combination of technical solutions and chemistry developments are, thus, a powerful combination. Specific radioactivity is a major concern in labelling synthesis. The search for methods that minimize isotopic dilution has been an important goal since the beginning of the 11C-chemistry era. The ratio of 11C/12C that now can be attained at end of synthesis is around 1/10,000, assuming a synthesis time of two half-lives. Obviously there is still much work be done, where miniaturization and closed on-line reaction apparatus are just two examples of methods that will surely further improve the specific radioactivity. The 11C-chemistry of today has reached a level where a few standard tracers are in widespread use for clinical investigations and clinical research. For basic research purposes there are numerous 11C-compounds available, and in the future some of them will probably be added to the standard tracer set-up at most major PET facilities. The Good Manufacturing Practice (GMP) concept should be the guideline for all tracer productions that are intended for human application. Processor-controlled automated synthetic devices should be used in the "manufacturing process", which would have the added effect of protecting the chemists performing the tracer productions from being exposed to radiation.

REFERENCES Alauddin MM, Ravert HT, Musachio JL, Mathews WB, Dannals RF and Conti P (1995) Selective alkylation of pyrimidyl dianions III: no-carrier-added synthesis of [11C-methyl]-thymidine. Nucl. Med. Bioi. 22, 791-795. Alauddin MM, Fissekis JD and Conti PS (1997) a-Alkylation of amino acid derivatives: synthesis and chiral resolution of [11C]p-aminoisobutyric acid. Nucl. Med. Biol. 24, 771-775.

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Al-Qahtani MH and Pike VW (2000) Palladium(II)-mediated 11C-carbonylative coupling of diaryl-iodonium salts with organostannanes - a new, mild and rapid synthesis of aryl [11C]ketones. J. Chem. Soc., Perkin Trans. 1, 6, 1033-1036. Amokthari M, Andersen K, Ibazizene M, Gourand F, Dauphin F and Barre L (1995) Synthesis of [11C]LU29-66, a 5-HT2 receptor antagonist via phosgene. J. Labelled Compd. Radiopharm. 42, 437-446. Angelini G, Carnavaletti F, Margonelli A, Corsi G, Ragni P, Fazio F, Toodde S and Tinti O (1999) Mild synthesis of [N-methyl-11C]-isovaleroyl-(L)-carnitine the usefulness of a tritium approach. Appl. Radiat. Isot. 50, 303-310. Andersson Y and Langstrom B (1994) Transition metal mediated reactions using [11C]cyanide in the synthesis of 11C-labelled aromatic compounds. J. Chem. Soc. Perkin Transact. 1395–1400. Andersson Y and Langstrom B (1995a) Synthesis of 11C-labelled ketones via carbonylative coupling reactions using [11C]carbon monoxide. J. Chem. Soc. Perkin. Trans. 1, 287-289. Andersson Y and Langstrom B (1995b) 11C-Methyl iodide and 11C-carbon monoxide in palladium promoted coupling reactions. J. Lab. Compd. Radiopharm. 37, 84–87. Andersson Y, Bergstrom M and Langstrom B (1994) Synthesis of 11C-labelled benzamide compounds as potential tracers for poly (ADP-ribose) synthetase. Appl. Radiat. Isot. 45, 707–714 Andersson Y, Cheng A and Langstrom B (1995) Palladium-promoted coupling reactions of 11 C-methyl iodide with organotin and organoboron compounds. Acta Chem Scand 49, 683-688. Andersson Y, Tyrefors N, Sihver S, Onoe H, Watanabe Y, Tsukada H and Langstrom B (1998) Synthesis of a 11C-labelled derivative of the N-methyl-D-aspartate receptor antagonist MK-801. J. Labelled Compd. Radiopharm. 49, 567-576. Antoni Gand Langstrom B (1986) Asymmetric synthesis of L-[3-11C]alanine. Acta Chem. Scand. B 40. 152156. Antoni G and Langstrom B (1987a) Synthesis of 3-11C-labelled alanine, 2-aminobutyric acid, norvaline, norleucine, leucine and phenylalanine and preparation of L-[3-11C]alanine and L-[311 C]phenylalanine. J. Labelled Compd. Radiopharm. 24, 125–143. Antoni G and Langstrom B (1987b) Asymmetric synthesis of L-2-amino[3-11C]butyric acid, L-[311 C]norvaline and L-[3-11C]valine. Acta Chem. Scand. B 41, 511–517. Antoni G and Langstrom B (1987c) Synthesis of DL-[3-11C]valine using [2-11C]isopropyl iodide, and preparation of L-[3-11C]valine by treatment with D-amino acid oxidase. Appl. Radiat. hot. 38, 655659. Antoni G and Langstrom B (1988) Synthesis of y-amino[4-11C]butyric acid (GABA) J. Labelled Compd. Radiopharm. 27, 571–576. Antoni G and Langstrom B (1992) Synthesis of 11C-labelled a,p-unsaturated nitriles. Appl. Radiat. hot. 43, 903-905. Antoni G, Ulin J and Langstrom B (1989) Synthesis of the 11C-labelled p-adrenergic receptor ligands atenolol, metoprolol and propanolol. Appl. Radiat. hot. 40, 561–564. Antoni G, Omura H, Bergstrom M, Furuya Y, Moulder R, Roberto A, Sundin A, Watanabe Y and Langstrom B (1997) Synthesis of L-2,4-diamino[4-11C]butyric acid and its use in some in vitro and in vivo tumour models. Nucl. Med. Biol. 24, 595–601.

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Antoni G. Omura H, Ikemoto M, Moulder R, Watanabe Y and Langstrom B (2001) Enzyme catalysed synthesis of L-[4-11C]aspartate and L-[5-11C]glutamate. J. Labelled Compd. Radiopharm. 44, 285294. Aquilonius SM, Bergstrom K, Eckernas SA, Hartvig P, Leenders KL, Lundqvist H, Antoni G, Gee A, Rimland A, Uhlin J and Langstrom B (1987) In vivo evaluation of striatal dopamine reuptake sites using 11C-nomifensine and positron emission tomography. Acta Neurol. Scand. 76, 282-287. Axelsson S, Langstrom B and Matsson O (1987) A new carbon isotope effect as a mechanistic probe determination of 11C/14C kinetic isotope effects in N-alkylations of tertiary amines. J. Am. Chem. Soc. 109, 7233-7235. Axelsson S, Bjurling P, Langstrom B and Matsson O (1992) Kinetic isotope effecting enzyme mechanism studies. 11C/14C kinetic isotope effect of p-tyrosinase catalysed p-elimination from L-tyrosine. J. Am. Chem. Soc. 114, 1502-1503. Balatoni JA, Adams MJ and Hall LD (1989) Synthesis of 11C-labelled aromatics using aryl chromium tricarbonyl intermediates. J. Labelled Compd. Radiopharm. 27, 1429. Barre L, Debruyne D, Lasne MC, Gourand F, Bonvento G, Camsonne R, Moulin M and Baron JC (1992) Synthesis and regional rat brain distribution of [11C]MDL72222 a 5HT3 receptor antagonist. App. Radiat. Isot. 43, 509–516. Barre L, Lasne MC and Charbonneau P (1995) Synthesis of non-carrier added [N-methyl-11C]roxi1hromycme. J. Labelled Compd. Radiopharm. 36, 801–803. Barrio JR, Phelps ME, Huang SC, Keen RE and MacDonald NS (1982a) [1-11C]leucine and the principle of metabolic trapping for the tomographic measurement of cerebral synthesis in man. J. Labelled. Compd. Radiopharm. 19, 1271–1272. Barrio JR, Egbert JE, Henze A, Schelbert HR and Baumgartner FJ (1982b) L-[4-11C]aspartic acid: Enzymatic synthesis, myocardial uptake and metabolism. J. Med. Chem. 25, 93-96. Berger G, Maziere M, Knipper R, Prenant C and Comar D (1979) Automated synthesis of carbon-11 labelled radiopharmaceuticals: imipramine, chlorpromazine, nicotine and methionine. Int. J. Appl. Radiat. Isot. 30, 393-399. Berger G, Maziere M, Prenant C and Comar D (1980a) Synthesis of carbon-11 labelled acetone. Int. J. Appl. Radiat. Isot. 31, 577-578. Berger G.,Maziere B, Sastre J and Comar D (1980b) Carrier-free 11C-formaldehyde: An approach. J. Labelled Compd. Radiopharm. 17, 59-71. Berger G, MaziereM, Godot JM, Prenant C and Comar D (1981a) Synthesis of radioactive 11C molecules for medical research on a micro-scale. A theoretical and practical approach. J. Labelled Compd. Radiopharm. 18, 1649–1671. Berger G, Maziere M, Prenant C, Sastre J and Comar D (1981b) Synthesis of high specific activity 11C-17amethyltestosterone. Int. J. Appl. Radiat. Isot. 32, 811-815. Berger G, Maziere B, Prenant C, Sastre J, Syrota A and Comar D (1982) Synthesis of 11C-propranolol. J. Radioanal. Chem. 74, 301-306. Bergstrom M, Westerberg G, Kihlberg T and Langstrom B (1997) Synthesis of some 11C-labelled MAO-A inhibitors and their in vivo uptake kinetics in rhesus monkey. Nucl. Med. Biol. 24, 381-388.

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Bergstrom M, Juhlin C, Bonasera TA, Sundin A, Rastad J, Akerstrom G and Langstrom B (1999) PET imaging of adrenal cortical tumors with the 11 p-hydroxylase-tracer 11C-metomidate. J. Nucl. Med. 41, 275-282. Bergstrom M, Fasth K-J, Kilpatrick G, Ward P, Cable KM, Wipperman MD, Sutherland DR and Langstrom B (2000) Brain uptake and receptor binding of two (11C)labelled selective high affinity NK1antagonists, GR203040 and GR205171 - PET studies in rhesus monkey Neuropharmacology 39, 664670. Berridge MS, Cassidy EH and Bordeaux KG (1994) Preparation of [11C]triamcininolene acetonide via [11C]acetone. Appl. Radiat. Isot. 45, 91–96. Bida GT, Ruth TJ and Wolf AP (1978) Experimentally determined thick target yields for the 14N(p,a)11C reaction. Radiochim. Acta 27, 181-185. Bjurling P and Langstrom B (1989) Synthesis of 1- and 3-11C-labelled L-lactic acid using multi-enzymatic catalysis. J. Labelled Compd. Radiopharm. 38, 427-432. Bjurling P, Watanabe Y and Langstrom B (1988) The synthesis of [3-11C]pyruvic acid, a useful synthon, via an enzymatic route. Appl. Radiat. Isot 39, 627-630. Bjurling P, Watanabe Y, Tokushige M, Oda T and Langstrom B (1989) Syntheses of (5-11 C-labelled Ltryptophan and 5-hydroxytryptophan by using a multi-enzymatic route. J. Chem. Soc. Perkin. Trans. 1331-1334. Bjurling P, Watanabe Y, Oka S, Nagasawa T, Yamada H and Langstrom B (1990a) Multi-enzymatic synthesis of p-11C-labelled L-tyrosine and L-DOPA. Acta Chem. Scand. 44, 183–188. Bjurling P, Antoni G, Watanabe Y and Langstrom B (1990b) Enzymatic synthesis of carboxy-11C-labelled Ltyrosine, L-DOPA, L-tryptophan and 5-hydroxy-L-tryptophan. Acta Chem. Scand. 44, 178-182. Bjorkman M and Langstrom B (2000) Functionalisation of 11C-labelled olefins via a Heck coupling reaction. J.Chem. Soc., Perkin Trans. 1 3031-3034. Bjorkman M, Andersson Y, Doi H, Kato K, Suzuki M, Noyori R, Watanabe Y and Langstrom B (1998) Synthesis of 11C/13C-labelled prostacyclins. Acta Chem. Scand. 52, 635-640. Bjorkman M, Doi H, Resul B, Suzuzki M, Noyori R, Watanabe Y and Langstrom B (2000a) Synthesis of a 11 C-labelled prostaglandin F2« analogue using an improved method for Stille reactions with 11 [ C]methyl iodide. J. Labelled Compd. Radiopharm. 43, 1327-1334. Bjorkman M Kihlberg T and Langstrom B (2000b) [11C]Carbon monoxide in a palladium-promoted formylation reaction of aryl halides followed by a Wittig reaction. Manuscript in: Bjorkman M. Doctoral Thesis, Acta Universitatis Upsaliensis No. 564, Uppsala 2000. Bolster JM, Vaalburg W, Paans AM van Dijk TH, Elsinga PH, Ziljstra JB, Piers DA, Mulder NH, Woldring MG and Wynberg H (1986) Carbon-11 labelled tyrosine to study tumour metabolism by positron emission tomography (PET). Eur. J. Nucl. Med. 12, 321-324. Bonnot-Lours S, Crouzel C, Prenant C and Hinnen F (1993) Carbon-11 labelling of an inhibitor of acetylchlolinesterase: [11C]physostigmine. J. Labelled Compd. Radiopharm. 33, 277-284. Borght TV, Labar D, Pauwels S and Lambotte (1991) Production of [2-11C]thymidine for quantification of cellular proliferation with PET. Appl. Radiat. Isot. 42, 103–104.

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Brady F, Luthra SK, Brown G, Osman S, Harte RJA, Denny WA, Baguley BC, Jones T and Price PM (1997) Carbon-11 labelling of the antitumour agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and determination of plasma metabolites in man. Appl. Radial, hot. 48, 487-492. Casey L, Digenis GA, Wesner DA, Washburn LC, Chaney JE, Hayes RL and Callahan (1981) Preparation and preliminary tissue studies of optically active 11C-D- and L-phenylalanine. Int. J. Appl. Radiat. hot. 32, 325-330. Castro-Ibarra A, Weber K and Oberdorfer F (1997) Synthesis of 1, 3-disubstituted 6-methyl-2-[11C]uracil derivatives using 11C-labelled dialkylurea and acetic anhydride. J. Labelled Compd. Radiopharm. 40, 810-811. Chakraborty P, Gildersleeve DA, Toorongian SA, Kilboum MR, Schwaiger M and Wieland DM (1993a) Synthesis of [11C]epinephrine and other biogenic amines by direct methylation of normethyl precursors. J. Labelled Compd. Radiopharm. 32, 172–173. Chakraborty P, Gildersleeve DA, Toorongian SA, Kilbourn MR, Schwaiger M and Wieland DM (1993b) High yield synthesis of high specific activity R-(-)r11C]epinephrine for routine PET studies in humans, Nucl. Med. Biol. 20, 939-944. Chakraborty PK, Manger TJ, and Chugani HT (1997) The synthesis of no-carrier-added [11C]urea from [11C]carbon dioxide and application to [11C]uracil synthesis. Appl. Radiat. hot. 48, 619-621. Channing MA and Simpson N (1993) Radiosynthesis of [l-11C]polyhomoallylic fatty acids. J. Labelled Compd. Radiopharm. 33, 541-546. Charalambous A, Manger TJ and Kilbourn MR (1994) Synthesis of (N-[11C]methyl)Y-29794 a competitive inhibitor of prolyl endopeptidase. J. Labelled Compd. Radiopharm. 34, 499-504. Charalambous A. Tluczek L, Frey KA, Higgins DS, Greenmyre TJ and Kilbourn MR (1995) Synthesis and biological evaluation in mice of (2-[11C]methoxy)-6',7'-dihydrorotenol, a second generation rotenoid for marking mitochondrial complex I activity. Nucl. Med. Biol. 22, 491–496. Christrnan DR, Hoyte RM and Wolf AP (1970) Organic radiopharmaceuticals labelled with isotopes of short half-life L: 11C-1-dopamine hydrochloride. J. Nucl. Med. 11, 474-478. Christman DR, Crawford EJ, Finn RD, Friedkin M and Wolf AP (1971) Detection of DNA synthesis in intact organisms with positron emitting methyl 11C-thymidine. Proc. Natl. Acad. Sci. USA 69, 988-992. Christman DR, Finn RD, Karlstrom K and Wolf AP (1975) The production of ultra high specific activity 11Clabeled hydrogen cyanide, carbon dioxide, carbon monoxide and methane via the l4N(p,a)11C reaction. Int. J. Appl. Radiat. Isot. 26, 435-442. Cohen MB, Spolter L, Chang CC, Cook JS and MacDonald NS (1980) Enzymatic synthesis of 11C-pyruvic acid and 11C-L-lactic acid. Int. J. Appl. Radiat. Isot. 31, 45-49. Cohen MB, Spolter I, Chang CC, Behrendt D, Cooks J and MacDonald N (1982) The varying tissue distribution of L-glutamic acid labeled in three different sites. Int. J. Appl. Radiat. hot. 33, 613-617. Collman JP, Hegedus LS, Norton JR and Finke RG (1987) Principles and Applications of Organotransition Metal Chemistry, University Science Books, Mill Valley, CA. Comar D, Carton JC, Maziere M and Marazano C (1976) Labelling and metabolism of methionine-methyl11 C. Eur. J. Nucl. Med. 1, 11–14.

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Conti PS, Alauddin MM, Fissekis JR, Schmall B and Watanabe KA (1995) Synthesis of 2'-fluoro-5-[11C]methyl-1-p-D-arabinofuranosyluracil ([11C]-FMAU): a potential nucleoside analog for in vivo study of cellular proliferation with PET. Nucl. Med. Biol. 22, 783-789. Conway T and Diksic M (1988) Synthesis of "no-carrier-added" carbon-11 SarCNU: the sarcosinamide analog of the chemotherapeutic agent BCNU. J. Nucl. Med. 29, 1957–1960. Crane HR and Lauritsen CC (1934). Further experiments with artificially produced radioactive substances. Phys. Rev. 45, 497. Crouzel C and Syrota A (1990) The use of [11C]diazaomethane for labelling a calcium channel antagonist: PN 200-110 (Isrodipine). Appl. Radial. Isot. 41, 241–242. Crouzel C, Langstrom B, Pike VW and Coenen HH (1987a) Recommendations for a practical production of [11C]methyl iodide. Appl. Radial, hot. 38, 601–603. Crouzel C, Amno R and Fournier D (1987b) Synthesis of carbon-11 labelled diazomethane. Appl. Radiat. Isot. 38, 669-670. Crouzel C, Hinnen F and Maitre E (1995) Radiosynthesis of methyl and heptyl [11C]isocyanates from [11C]phosgene, application to the synthesis of carbamates: physostigmine and [11Qheptylphysostigmine. Appl. Radiat. Isot. 46, 167-170. Dannals RF, Ravert HT, Frost J, Wilson AA, Burns HD and Wagner HN (1985) Radiosynthesis of an opiate receptor binding radiotracen [11C]carfentanil. Int. J. Appl. Radiat. Isot. 36, 303. Dannals RF, Ravert HT, Wilson AA and Wagner HN (1990) Synthesis of a selective serotonin uptake inhibitor [11C]citalopram. Appl. Radiat. Isot. 41, 541–543. DaSilva JN, Schwartz RA, Greenwald ER, Lourenco CM, Wilson AA and Houle S (1999) Dopamine Dl agonist R-[11C]SKF82957: synthesis and in vivo characterization in rats. Nucl. Med. Biol. 26, 537542. DaSilva JN, Lourenco CM, Wilson AA and Houle S (2001) Syntheses of the phosphodiesterase-4 inhibitors [11C]Ro 20-1724, R, R/S- and 5-[11C]rolipram. J. Labelled Compd. Radiopharm. 44, 373-384. Davenport RJ, Pike VW, Dowsett K, Turton DR and Poole K (1997) Automated chemo-enzymatic synthesis of no-carrier-added [carbonyl-11C]propionyl-L-carnitine. Appl. Radiat. Isot. 48, 917-924. Delforge J, Janier M, Syrota A, Crouzel C, Vallois JM, Cayla J, Lancon JP and Mazoyer BM (1990) Noninvasive quantification of muscarinergic receptors in vivo with positron emission tomography in the dog heart. Circulation 82, 1494–1504. Delforge J, Syrota A, Lancon JP, Nakajima K, Loc'h C, Janier M, Vallois JM and Crouzel C (1991) Cardiac beta-adrenergic receptor density measured in vivo using PET, CGP12177, and a new graphical method. J. Nucl. Med. 32, 739-748. Dence CS, Napolitano E, Katzenellenbogen JA and Welch MJ (1994) Carbon-11-labeled estrogens as potential imaging agents for breast tumours. Nucl. Med. Biol. 23, 491 -496. De Rosario RB, Jung YW, Caraher J, Chakraborty PK and Wieland DM (19%) Synthesis and preliminary evaluation of [11C]-(-)-phenylephrine as a functional heart neuronal PET agent. Nucl. Med. Biol. 23, 611-616. de Groot T, Manis B, Boonen C, Borman G, Mortelmans L, Eisenhut N and Verbruggen A (1997) Evaluation of carbon-11-labelled phenoxy-bridged fatty acids for studying myocardial fatty acid metabolism with PET. Nucl. Med. Biol. 24, 461-464.

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Nagren K, Ragnarsson U and Langstrom B (1986) The synthesis of the neuropeptide met-enkephalin and two metabolic fragments labelled with 11C in the methionine group. Appl. Radiat. Isot. 37, 537-539. Nagren K, Ragnarsson U, and Langstrom B (1988) Synthesis of three 11C-labelled methionine-containing enkephalin analogues. J. Labelled Compd. Radiopharm. 25, 149-160. Nagren K, Takahashi T, Lehikoinen P and Bergman K (1991) Preparation of the antioestrogenic compound N-[methyl- 11C]toremifene for the study of oestrogen-receptor positive tumours in vivo. J. Labelled Compd. Radiopharm. 29, 1085-1089. Nagren K, Schoeps KO, Halldin C, Swahn CG and Farde L (1994) Selective synthesis of racemic 1-11C-labelled norepinephrine, octopamine, norphenylephrine and phenethanolamine using [11C]nitromethane. Appl. Radiat. Isot. 45,515-522. Nagren K, Muller L, Halldin C, Swahn CG and Leuhkoinen P (1995) Improved synthesis of some commonly used PET radioligands by the use of [11C]methyl triflate. Nucl. Med. Biol. 22, 254-239). Nagren K, Halldin C, Swahn CG, Suhara T and Farde L (1996) [11C]Metaraminol, a false neurotransmitter preparation, metabolite studies and positron emission tomography examination in monkey. Nucl. Med. Biol. 23, 221-227. Neu H, Kihlberg T and Langstrom B (1997a) Synthesis of saturated fatty acids 11C (13C)-labelled in the G> methyl position. J. Labelled Compd. Radiopharm. 39, 509-524. Neu H, Hartvig P, Torstenson R, Fasth KJ, Sonesson C, Waters N, Carlsson A, Tedroff J and Langstrom B (1997b) Synthesis of [11C]methyl-(-)-OSU6162, its regional brain distribution and some pharmacological effects of (-)-OSU6162 in the dopaminergic system studied in the rhesus monkey by positron emission tomography. Nucl. Med. Biol. 24, 502–511. Neu H, Bonasera T and Langstrom B (1998) Lithium [11C]methyl(2-thienyl)cuprate LiCN in 1,4-additions to a,p-unsaturated ketones. 11C/13C-labelling of the androgen mesterolone. J. Labelled Compd Radiopharm. 41, 227-235. Niisawa K, Ogawa K, Saio J., Taki K, Karazawa T and Nozaki T (1984) Production of no-carrier-added 11Ccarbon disulfide and 11C-hydrogen cyanide by microwave discharge. Int. J. Appl. Radiat. hot. 35, 2933. Nishiyama S, Sato K, Harada N, Kakiuchi T and Tsukada H (2000) Development and evaluation of muscarinic cholinergic receptor ligands N-[11C]ethyl-4-piperidyl benzilate and N-[11C]propyl-4piperidyl benzilate: A PET study in comparison with N-[11C]methyl-4-piperidyl benzilate in the conscious monkey brain. Nucl. Med. Biol. 27, 733.740. Oberdorfer F, Siegel T, Guhlman A, Keppler D and Maier-Borst W (1992) The preparation of a 11C-labelled 5lipoxygenas product. 5(S)-hydroxy-6XRHN-[1-11C]acetyl)cistinyl-7,9-rr<3rj5-ll,14-a'5-eicosatetraenoic acid. J. Labelled Compd. Radiopharm. 31, 903–913. Oberdorfer F, Zobeley A, Weber K, Prenant C, Haberkorn U and Mair-Borst W (1993) Preparation of a-[311 C]aminoisobutyric acid from an azadisilolidine derivative of alanine. J. Labelled Compd. Radiopharm. 33, 345-353. Oberdorfer F, Theobald A and Prenant C (1996) Simple production of [1-carbon-11]acetate. J. Nucl. Med. 37, 341–342. Ogawa K, Sasaki M and Nozaki T (1997) Malonic esters and acetoacetic ester synthesis of 2-[11, 14 C]methylfatty acids. Appl. Radiat. Isot. 48, 623-630.

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Ogren M and Langstrom B (1997a) Synthesis of 2-deoxy-D-[1-11C]glucose. In Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 292, ISBN 91-554-4000-2 Ogren M and Langstrom B (1998) Synthesis of D-[1-11C]mannitol and the enzymatic oxidation to D-[111

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Reivich M, Alavi A, Wolf AP, Greenberg JH, Fowler JS, Christman DR, MacGregor R, Jones SC, London J, Shiue C and Yonekura Y (1982) Use of 2-deoxy-D[1-11C]glucose for the determination of local cerebral glucose metabolism in humans. Variations within and between subjects. J. Cereb. Blood Flow. Metab. 2, 307-319. Roeda D and Westera G (1981) A UV-induced on-line synthesis of 11C-phosgene and the preparation of some of its derivatives. Int. J. Appl. Radial. Isot. 32, 931–932. Roeda D and Crouzel (2001) [11C]Formaldehyde revisited: considerable concurrent [11C]formic acid formation in the low-temperature conversion of [11C]carbon dioxide into [11C]formaldehyde Appl. Radiat. Isot. 54, 935-939. Roeda D, Crouzel C and van Zanten B (1978) The production of 1lC-phosgene without added carrier. Radiochem. Radioanal. Lett. 33, 175–178. Roeda D, Crouzel C, Brouillet E and Valette H (1996) Synthesis and in vivo distribution of no-carrier-added N(i))-nitro-L-arginine[11C]methyl ester, as a nitric oxide synthase inhibitor. Nucl. Med. Biol. 23, 509512. Roeda D, Bramoulle Y, Richards C, Jegou J, Dolle F and Crouzel C (1999a) Towards a carbon-11-labelled trifluoroacetyl substituent on an aromatic ring. The new precursor [11C]trifluoroacetomtrile. J. Labelled Compd. Radiopharm. 42, S119-S120. Roeda D, Bramoulle Y, Richard C, Dolle F and Crouzel C (1999b) Carbon-11 labelled trimethylsilylbenzenes from [11C]methyllithium or [11C]trimethylsilyl chloride - application to zifrosilon. J. Labelled Compd. Radiopharm. 42, 126–S128. Roeda D, Dolle F and Crouzel C(1997) Synthesis of 4-substituted [2-11C]imidazoles from [11C]formaldehyde. J. Labelled Compd. Radiopharm. 40, 725-726. Ropchan JR and Barrio JR (1984) Enzymatic synthesis of [1-11C]pyruvic acid, L-[1-11C]lactic acid and L-[1[11C]alanine via D,L-[1-11C]alanine. J. Nucl. Med. 25, 887-892. Rosenspire KC, Haka MS, Van Dort ME, Jewett DM, Gildersleeve DL, Schwaiger M, Massin CC and Wieland DM (1990) Synthesis of and preliminary evaluation of carbon-11-meta-hydroxyephedrine: a false transmitter agent for heart neuronal imaging. J. Nucl. Med. 31, 1328–1334. Ruben S, Hassid WZ and Kamen MD (1939) Reactive carbon in the study of photosynthesis. J. Am. Chem. Soc.61, 661. Samson Y., Hantraye P., Baron J.C., Soussaline F., Comar D. and Maziere M. (1985) Kinetics and displacement of [Ro 15-1788, a benzodiazepine anatagonist, studied in human brain in vivo by positron emission tomography. Eur. J. Pharmacol. 110, 247–251. Sandell J, Halldin C, Pike VW, Chou YH, Varnas K, Hall H, Marchais S, Nowicki B, Wikstrom H, Swahn CG and Farde L (2001) New halogenated [11C]WAY analogues, [11C]6FPWAY and [11C]6BPWAY radiosynthesis and assessment as radioligands for the study of brain 5-HT1A receptors in living monkey. Nucl. Med. Biol. 28, 177-185. Sasaki T, Tokyama T, Oda K, Toyama H, Ishii SI, Senda M, Karasawa K, Satoh N, Setaka M, Nojima S, Nozaki T and Braquet P (1996) A distribution study of 11C-platelet-activating factor (PAF) analogs in normal and tumour bearing mice. Nucl. Med. Biol. 23, 309–314.

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Sasaki M, Ikemoto M, Mutoh M, Haradahira T, Tanaka A, Watanabe Y and Suzuki K (1999) Automated synthesis of L-[p-11C]amino acids using an immobilized enzyme column. Appl. Radiat. Isot. 52, 199– 204. Sassaman MB, Danico M, Schmall B and Eckelman WC (1999) [11C]Diazepam synthesis: N[11C]methylation of desmethyldiazepam is facilitated by utilization of a preformed sodium salt/benzo15-crown-5 complex. J. Labelled Compd. Radiopharm. 42, 1229–1233. Schirbel A, Holsbach MH and Coenen HH (1999) N.C.A. [11C]CO2 as a safe substitute for phosgene in the carbonylation of primary amines. J. Labelled Compd. Radiopharm. 42, 537–551. Schmall B, Conti S and Alauddin MM (1996) Synthesis of [11C-methyl]-a-aminoisobutyric acid (AIB). Nucl. Med. Biol. 23, 263-266. Smith CB, Davidsen L, Deibler G, Patlak C, Petigrew K and Sokoloff L (1980) A method for the determination of local rates of protein synthesis in man. Trans. Am. Soc. Neurochem. 11, 94. Schmitz F, Plenevaux A, Del-Fiore G, Lemaire C, Comar D and Luxen A (1995) Fast routine production of L-[11C-methyl]methionine with A12O3/KF. Appl. Radiat. hot. 46, 893-898. Schoeps KO and Halldin C (1992) Synthesis of racemic [a-11C]amphetamine and [a-11C]phenethylamine from [11C]nitroalkanes. J. Labelled Compd. Radiopharm. 31, 891–902. Schoeps KO, Halldin C, Nagren K, Swahn CG, Karlsson P Hall H and Farde L (1993) Preparation of [1[11C]dopamaine,[1-11C]p-tyramine and [1-11C]m-tyramine, autoradiography and PET examinations of [1-11C]dopamine in primates. Appl. Radiat. Isot. 20, 669.678. Schoeps KO, Stone-Elander S and Halldin C (1989) On-line synthesis of [11C]nitroalkanes. Appl. Radiat. Isot. 40, 261–267. Schoeps KO, Langstrom B, Stone-Elander S and Halldin C (1991) Synthesis of [1-11C]D-glucose and [111 C]mannose from on-line produced [11C]nitromethane. Appl. Radiat. Isot. 42, 877-882. Shah F, Pike VW, Dowsett K and Aigbirhio FI (1994a) Approaches to the synthesis of [11C]olefins and derivatives as novel labelling agents. J. Labelled Compd. Radiopharm. 35, 83-85. Shah F, Hume SP, Pike VW, Ashworth S and McDermott J (1994b) Synthesis of the enantiomers of [Nmethyl-11C]PK11195 and comparison of their behaviour as radioligands for PK binding sites in rat. Nucl. Med. Biol. 21, 573–581. Shah F, Pike VW and Dowsett K (1997) Preparation of no-carrier-added [1-11C]ethylene and [1-11C]1,2dibromoethane as new labelling agents. Appl. Radiat. Isot. 48, 931-941. Shi B, Tanjore K, Narayanan K, Yang ZY, Bradley TC and Mukherje J (1999) Radiosynthesis of 2-(N-alkyl1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors. Nucl. Med. Biol. 26, 725-735. Shiue CY and Wolf AP (1981) The synthesis of [1-11C]-D-glucose for the measurement of brain glucose metabolism. J. Nucl. Med. 22, 58. Sihver W, Fasth KJ, Ogren M, Lundkvist H, Bergstrom M, Langstrom B and Nordberg A (1999) In vivo positron emission tomography studies on the novel nicotinic receptor agonist [11C]MPA compared with [11C]ABT-418 and (S)-(-)[11C]nicotine in rhesus monkey. Nucl. Med. Biol. 26, 633-640. Snyder SE, Tluczek L, Jewett DM, Nguyen TB, Kuhl DE and Kilbourn R (1999a) Synthesis of 1[11C]methylpiperidine-4-yl propionate ([11C]PMP) for in vivo measurements of acetylcholinesterase activity. Nucl. Med. Biol. 25, 751–754.

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Snyder SE, Sherman PS, Desmond TJ, Frey KA and Kilbourn MR (1999b) (-)-6',7'-[11C]dihydroroten-12a01 ((-)-[11C]DHROL) for in vivo measurement of mitochondrial complex I. J. Labelled Compd. Radiopharm. 42, 641-652. Soussain R, Gueguen P, Morgat JL, Maziere B, Berger G and Comar D (1984) Enzymatic synthesis of 11Clabelled (-)-epinephrine. J. Labelled Compd. Radiopharm. 21, 203-209. Spang JE, Patt JT, Westera G and Schubiger PA (2000) Comparison of N[11C]methylnorchloroepibatidine and N[11C]methyl-2-(2-pyridyl)-7-azabicyclo(2.2.1)-heptane with N-[11C]methyl-epibatidine in small animal PET studies. Nucl. Med. Biol. 27, 239-247. Spang JE, Patt JT, Westera G and Schubiger PA (1999) Synthesis and [11C]-radiolabelling of dechloroepibatidine and 2PABH, two potential radioligands for studying the central nAchRs in vivo. J. Labelled Compd. Radiopharm. 42, 761–771. Spolter L, Cohen MB, Chang CC and McDonald NS (1979) Enzymatic synthesis of C-11 acetyl carnitine. J. Nucl. Med. 20, 662-663. Steel CJ, Brown GD, Dowsett K, Turton DR, Luthra SK, Tochan-Danguy H., Waters SL, Price P and Brady F (1993) Synthesis of 2-[11C]thymine from [11C]phosgene: a precursor for 2-[11C]thymidine. J. Labelled Compd. Radiopharm. 32, 178–179. Steinbach J, Mading P, Fuchtner F and Johannsen B (1995) N.C.A. 11C-labelling of benzenoid compounds in ring positions: synthesis of nitro-[1-11C]benzene and [1-11C]aniline. J. Labelled Compd. Radiopharm. 36, 33-42. Stille JK (1986) The palladium-catalyzed cross-coupling reactions of organotin reagents with organic electrophiles. Angew. Chem., Int. Ed. Engl. 25, 508-524. Straatman MG and Welch MJ (1974) A general method for labelling proteins with 11C. J. Nucl. Med. 16, 425-428. Stone-Elander S, Elander N, Thorell JO, Sola's G and Svennebrink J (1994) A single-mode microwave cavity for reducing radiolabelling reaction times, demonstrated by alkylation with [[11C]C]alkyl halides. J. Labelled Compd. Radiopharm. 34, 949-960. Suchiro M, Scheffel U, Ravert HT, Dannals RF and Wagner HN (1993) [[11C]McCN5652 as a radiotracer for imaging uptake sites with PET. Life Sciences 53, 883-892. Suchiro M, Ravert HT, Wilson AA, Scheffel U, Dannals RF and Wagner HN (1992) Further investigation on the radiosynthesis of L-[11C]methyl-tryptophan. J. Labelled Compd. Radiopharm. 31, 151-157. Suehiro M, Wang TS, Yatabe T, van Heertum RL and Mann JJ (1998) Synthesis of [11C] and [3H]LY274601, a serotoninIA receptor agonist. J. Labelled Compd. Radiopharm. 41, 725–730. Suzuki A (1991) Synthetic studies via the cross-coupling reaction of organ and boron derivatives with organic halides. Pure & Appl. Chem. 63, 419. Suzuki M, Doi H, Kato K, Bjorkman M, Langstrom B, Watanabe Y and Noyori R (2000) Rapid methylation for the synthesis of a 11C-labelled tolylisocarbacyclin imaging the IP2 receptor in a living human brain. Tetrahedon 56, 8263–8273. Svard H, Nagren K, Malmborg P, Sohn D, Sjoberg S and Langstrom B (1982) The synthesis of various N[11C-methyl]-pharmaceuticals using 11C-methyl iodide. J. Labelled Compd. Radiopharm. 19, 1519520.

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Svard H, Jigerius SB and Langstrom B (1990) The Enzymatic synthesis of L-3 11C-serine. Appl. Radiat. Isotop. A 41, 587–591. Syrota A (1991) PET measurements of postsynaptic muscarinic and beta-adrenergic receptors in the heart. In: In vivo Imaging of Neurotransmitte Functions in Brain, Heart and Tumours. Kuhl DE (ed) The American College of Nuclear Physicians, Washingtons D.C 363–392. Syrota A and Merlet P (1996) Positron emission tomography: evaluation of cardiac receptors and neuronal functions. In: Cardiac Imaging: A Companion to Braunwald's Heart Disease. 2nd ed. Skorton DJ, (ed) W.B. Saunders Company, Philadelphia 1186–1203. Tada M, Oikawa H, Matzuzawa T, Itoh M, Fukuda H, Kubota K, Kawai H, Abe Y, Ugiyama H, Ido T, Ishiwata K, Iwata R, Imahori Y and Sato S (1989) A convenient synthesis of D-[1-11C]glucopyronose and D-[1-11C]galactopyranose. J. Labelled Compd. Radiopharm. 27, 1–8. Tang DY, Lipman A, Meyer GJ, Wan CN and Wolf AP (1979) 11C-Labelled octanal and benzaldehyde. J. Labelled Compd. Radiopharm. 16, 435-440. Takahashi K, Murakami M, Miura S, Lida H, Kanno I and Vemura K (1999) Synthesis and autoradiographic localization of muscarinic cholinergic antagonist (+)N-[11C]ethyl-3-piperidyl benzilate as a potent radioligand for positron emission tomography. Appl. Radiat. Isot. 50, 521–525. Takahashi T, Nagren K and Aho K (1990) An alternative synthesis of DL-[1-11C]alanine from [11C]HCN. Appl. Radiat. Isot. 41, 1187–1191. Tavitian B, Pappata S, Bonnot-Lours S, Prenant C, Jobert A, Crouzel C and Di Giamberardino L (1993) Positron emission tomography study of [11C]methyl-tetrahydroaminoacridine (methyl-tacrine) in baboon brain. Eur. J. Pharmacol. 236, 229-238. Tedroff J, Aquilonius SM, Hartvig P, Lundqvist H, Bjurling P and Langstrom B (1992) Estimation of regional cerebral utilization of [11C]3,4-dihydroxyphenylalanine (DOPA) in the primate by positron emission tomography. Acta Neurol. Scand. 85, 166-173. Tewson TJ, Fransceshini M, Gillum K, Kinsey B, Tilbury R and Berridge M (1989) The synthesis of [1[11C]pyruvic acid. J. Nucl. Med. 30, 928. Thorell JO, Stone-Elander S and Blander N (1992) Use of a microwave cavity to reduce reaction times in radiolabelling with [11C]cyanide. J. Labelled. Compd. Radiopharm. 31, 207–218. Thorell JO, Stone-Elander S, Hoist H and Ingvar M (1993a) Synthesis of [1-11C]D-glucosamine and evaluation of its in vivo distribution in rat with PET. Appl. Radiat. Isot. 44, 799-805. Thorell JO, Stone-Elander S and Elander N (1993b) Preparation of [11C]diethyloxalate and [11C]oxalic acid and demonstration of their use in the synthesis of [11C]-2,3,-dihydroxyquinoxaline. J. Labelled Compd. Radiopharm. 33, 995–1005. Thorell JO, Stone-Elander S and Blander N (1994) Difunctional two-carbon molecules from [11C]cyanide. J. Labelled Compd. Radiopharm. 34, 383-390. Thorell JO, Stone-Elander S, Ingvar M and Eriksson L (1995) Synthesis of [2-11C]-6,7-dihydroxyquinoxaline and evaluation of its in vivo distribution in rats with PET. J. Labelled Compd. Radiopharm. 36, 251257. Thorell JO, Stone-Elander S, Duelfer T, Cai SX, Jones L, Pfefferkorn H and Ciszewska G (1998) Synthesis of a 11C-labelled nitrated l,4-dihydroxyquinoxaline-2,3-dione the NMDA glycine receptor antagonist ACEA 1021 (Licostinel). J. Labelled Compd. Radiopharm. 41, 345-353.

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Tobias CA, Lawrence JH, Roughton FJW, Root WS and Gregersen MI (1945) The elimination of carbon monoxide from the human body with reference to the possible conversion of CO to CO2. Amer. J, Physiol, 145, 253. Turton DR, Brady F, Pike VW, Selwyn AP, Shea MJ, Wilson RA and De Landshere (1984) Preparation of human serum [methyl- 11C]methylalbumin microspheres and serum [methyl-11C]methylalbumin for clinical use. Int. J. Appl. Radiat. Isot. 35, 337. Ulin J, Gee AD, Malmborg P, Tedroff J and Langstrdm B (1989) Synthesis of racemic, (+) and (-) N[methyl- 11C]nomifensine, a ligand for evaluation of monoamine re-uptake sites by use of positron emission tomography. Appl. Radiat. hot. 40, 171–176. Vaalburg W, Beerling-van der Molen HD, Reiffers S, Rijskamp A, Woldring MG and Wynberg H (1976) Preparation of carbon-11 labeled phenylalanine and phenylglycine by a new amino acid synthesis. Int. J. Appl. Radiat. Isot. 27, 153–157. Vandersteene I, Andenaert K, Siegers G and Dierck RA (1998) Synthesis of [11C]granisetron, a possible positron emission tomography ligand for 5-HT3 receptor studies. J. Labelled Compd. Radiopharm. 41, 371-180. Vedejs E, Haight AR and Moss WO (1992) Internal coordination at tin promotes selective alkyl transfer in the still coupling reacion. J. Am. Chem. Soc. 114, 6556-6558. Volkow ND, Fowler JS, Gatley J, Logan J, Wang GJ, Ding YS and Dewey S (1996) PET evaluation of the dopaminergic system of the human brain. J. Nucl. Med. 37, 1242–1256. Wagner R, Stocklin G and Schaak W, (1981) Production of carbon-11 labelled methyl iodide by direct recoil synthesis. J. Labelled Compd. Radiopharm. 18, 1557-1566. Wagner HN, Burns HD, Dannals RF, Wonf DF, Langstrom B, Duelfers T, Frost JJ, Ravert HT, Links JM, Rosenbloom SB, Lukas SE, Kramer AV and Kuhar MJ (1983) Imaging dopamine receptors in the human brain by positron emission tomography. Science 221, 1264–1266. Wang WF, Ishiwata K, Nonaka H, Ishii SI, Kiyosawa M, Shimada JI, Suzuku F, and Senda M (2000) Carbon-11 labelled KF21213: A highly selective ligand for mapping CNS adenosine A2A receptors with positron emission tomography. Nucl. Med. Biol. 27, 541–546. Washburn LC, Sun TT, Byrd BL and Callahan AP (1982) Production of L-[1-11C]valine by HPLC resolution. J. Nuc. Med. 23, 29–33. Westerberg G and Langstrom B (1993) Synthesis of [11C]- and (13C)-cyanogen bromide, useful electrophilic labeling precursor. Acta Chem. Scand. 47, 974-978. Westerberg G and Langstrom B (1994a) Synthesis of sodium [11C]thiocyanate using [11C]cyanogens bromide. J. Labelled Compd. Radiopharm. 34, 545-548. Westerberg G and Langstrom B (1994b) Labelling of proteins with 11C in high specific radioactivity: [11C]albumin and [11C]transferrin. Appl. Radiat. Isot. 45, 773–782. Westerberg G, and Langstrom B (1997a) On-line producion of [11C]cyanogenbromide. App. Radiat. Isot. 48, 459–461. Westerberg G and Langstrom B (1997b) Synthesis of meta-iodobenzyl [11C]guanidine. J. Labelled Compd. Radiopharm. 39, 525-529. Westerberg G, Karcher W, Onoe H and Langstrom B (1994) [11C]Cyanogen bromide in the synthesis of 1,3di(tolyl)-[11C]guanidine. J. Labelled Compd. Radiopharm. 34, 691-696.

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6. CHEMISTRY OF FLUORINE-18 RADIOPHARMACEUTICALS SCOTT E. SNYDER AND MICHAEL R. KILBOURN Division of Nuclear Medicine, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI, USA.

INTRODUCTION The element fluorine is in many ways unique, both in chemical characteristics and usefulness in the pharmaceutical and chemical industries. Fluorine has a very small steric size and exhibits very high carbonfluorine bond energies; as fluorine is also extremely electronegative, such substitution can often produce significant and useful changes in physiochemical and biological properties of organic compounds. In some cases, substitution with fluorine produces a derivative with improved pharmacological properties. Although sometimes considered as an isosteric replacement for hydrogen, the differences in electronegativity and hydrogen bonding capability of fluorine often make it more like a substitution with a hydroxyl group. Many polyfluorinated organic compounds exhibit unusual and valuable properties (e.g., Teflon and Freons). The substitution of organic pharmaceuticals with fluorine has a long and successful history, and is currently widely practiced. Fluorine-18 is no less a remarkable and versatile positron-emitting radionuclide. Decay of fluorine-18 is largely by positron emission (97%), with a relatively low energy (maximum 0.635 MeV) and thus the emitted positron has a short mean range (2.39 mm in water). Fluorine-18 is readily available from both particle accelerators and nuclear reactors, using a wide variety of nuclear reactions, and can be produced at specific activities approaching the theoretical limit of 1.71 x 109 Ci/mmol. The moderate length half-life of 109.7 minutes allows for considerable latitude in the synthesis of radiopharmaceuticals: using a rule of thumb of limiting synthesis time to no more than three half-lives of a radionuclide, this would allow synthesis times as long as nearly six hours, although in practice such lengthy procedures would be the exception. The relatively long half-life of fluorine (in comparison, for example, to carbon-11, oxygen-15 and nitrogen-13) permits centralized syntheses of fluorine-18 labeled radiopharmaceuticals and distribution to locations distant from the cyclotrons which produced the radionuclide. As noted above, fluorine is disproportionately found in many pharmaceuticals, providing a rich assortment of structures and drugs amenable to labeling with fluorine-18. Finally, the longer half-life also provides opportunities for extended imaging protocols, sometimes continuing as much as 6 to 10 hours. Although discovered as early as 1937, synthetic applications of fluorine-18 for many years lagged behind radiochemlcal applications of carbon-11, largely due to the difficulties (sometimes only perceived) in fluorination of organic molecules. The majority of the methods for labeling with fluorine-18 have thus been developed or greatly improved in the last two decades. Similarly, most of the fluorine-18 labeled

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radiopharmaceuticals utilized for human studies have also been introduced in the last 20 years. The field of fluorine-18 chemistry has greatly expanded in only the last ten years, making it impossible to provide here a more comprehensive review such as that published in 1990 (Kilboum, 1990); that review had over four hundred references, and for the intervening decade alone there are well over two thousand literature citations for fluorine-18. Fortunately, along with this explosion in information have come excellent and widely available methods for searching computer databases of the primary scientific literature. Therefore, no attempt will be made here to provide a comprehensive literature review of the field of fluorine-18 chemistry or the synthesis of fluorine-18 labeled radiopharmaceuticals. Instead, this chapter will discuss some of the past and current issues and problems surrounding the practical methods currently in widespread use for production of fluorine-18 and labeling of radiopharmaceuticals, followed by short discussions of the radiopharmaceuticals currently (or which may soon be) in clinical use for research or patient care. PRODUCTION OF FLUORINE-18 Historically, a variety of cyclotron, linear-accelerator and reactor methods have been used to produce fluorine-18 for chemical or medical purposes (Table 1). For all practical purposes nearly all fluorine-18 is currently produced using a single nuclear reaction, 18O(p,n)18F, with the majority of the radionuclide isolated as the [18F]fluoride ion in aqueous solution. Specific activities of the radionuclide are very high (as much as 7400 TBq/mmol (2 x 105 Ci/mmol)), and the use of appropriate cyclotron targetry allows batch production of several curies of fluorine-18 in a single irradiation. The design of cyclotron targets for efficient irradiation of oxygen-18 water has been repeatedly discussed over the years, but in the end many of the targets are quite similar: all are constructed from an inert metal such as silver, titanium or tantalum, designed to withstand moderate to high internal pressures, and with small internal volumes to minimize the cost of the oxygen-18 enriched water. Table 1. Nuclear reactions used to prepare fluorine-18 using accelerators or reactors. 20

Ne(d,ct)18F 20 Ne(p,2pn)18F 16 O(3He,p)18F 16 O(a,pn)18F 18 O(p,n)18F 20 Ne(3He,n)18Ne,18Ne -18F 6 Li(n,a)3H,16O(3H,n)18F Use of the p,n reaction on an oxygen-18 enriched oxygen gas target can also be used to produce fluorine-18 labeled fluorine gas, by a two-step procedure involving first irradiation to form the radionuclide which attaches (by means not quite clear) to the inside target surface, followed by irradiation in the presence of a small amount of carrier fluorine in an inert gas. By this method [18F]F2 of moderate specific activity can be obtained, suitable for electrophilic fluorination reactions.

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Much more limited use is today made of the deuteron irradiation of neon gas (20Ne(d,a)18F), and generally only for the production of labeled fluorine gas ([18F]F2). Whereas twenty years ago this reaction was still employed for production of [18F]fluoride ion, today that has been totally supplanted by the 18O(p,n)18F reaction on oxygen-18 enriched water as discussed above. Production of fluorine-18 gas via the 20Ne(d,a) 18F nuclear reaction is necessarily a carrier-added method, as a small amount of fluorine gas must be introduced into the target to allow recovery of the radionuclide. The other methods for fluorine-18 production listed in Table 1 are rarely if ever used today, and production methods using these nuclear reactions have not changed significantly during the last decade. METHODS FOR [18F]FLUORINATION The chemical reactions used for labeling of organic molecules with fluorine-18 fall, in broad terms, into two categories: nucleophilic and electrophilic reactions. This division is based on the use of a nucleophilic form of fluorine-18 ([18F]fluoride ion) or an electrophilic form ([18F]F2), although there are some reactions which utilize nucleophilic fluoride ion in the presence of an electrophilic reagent, and thus accomplish an unusual combination of the two strategies. Historically, many important fluorine-18 labeled radiopharmaceuticals were initially prepared using electrophilic fluorination and subsequently higher yielding, higher specific activity nucleophilic routes of synthesis developed. NUCLEOPHILIC FLUORINATION [18F]Fluoride ion is almost always obtained as an aqueous solution, most often as a product of direct irradiation of a [18O]water target, although in rarer cases by using water to elute the radionuclide off the walls of an [18O]oxygen gas target. In aqueous form [18F]fluoride ion is quite unreactive, and requires some simple but very important manipulations to provide a reactive nucleophilic reagent. Many years of effort were expended in devising, evaluating and perfecting methods for preparation of reactive [18F]fluoride ion in organic solvents suitable for chemical syntheses. Although some of these problems from a decade ago persist, most have been relegated to a minor status, not often encountered in today's radiopharmaceutical laboratories. As the steps in preparing reactive [18F]fluoride ion are crucial to the subsequent success of the radiolabeling reactions, it is worth a quick glance at the steps involved in preparing the reagent. Adding a cation. Following irradiation of a [18O]water target, the [18F]fluoride ion must be accompanied by a positively charged counterion, but in all cases the identity of these ions has remained unclear. As a consequence of irradiating the [18O]water in metal targets there is very likely presence of metal ions representing the composition of the target body or the target foil (or perhaps metal ions present in the target water before irradiation). However, these metal ions have been found to be insufficient either in quantity or composition to provide sufficiently reactive [18F]fluoride ion by mere evaporation of the water. This problem is very effectively solved by the addition of a cationic counterion prior to the evaporation of the water. Three types of counterions have been used: large but soft metal ions such as rubidium or cesium, potassium complexed by a cryptand such as Kryptofix 222, or tetraalkylammonium salts. Through the years the larger metal ions such as cesium and rubidium have fallen out of favor. Many published syntheses now utilize the potassium/Kryptofix system, although examples of the use of tetraalkylammonium salts persist.

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The addition of a cation does, of course, also involve addition of another anion to the reaction mixture. Nonnucleophilic anions, such as hydroxide or carbonate, are utilized as they do not effectively compete with the [18F]fluoride ion in nucleophilic displacement reactions. The basicity of these added anions needs to be considered, however; in particular hydroxide ion in an aprotic solvent can cause unwanted base-catalyzed side reactions, and for this reason the carbonate ion is often chosen. Removing the water. Fluoride ion in water is quite unreactive. The evaporation of water is fairly straightforward; applications of heat and use of a solvent such as acetonitrile to provide a lower boiling azeotrope provide an efficient method to obtain reasonably anhydrous and reactive [18F]fluoride ion. The procedures have become so reliable and simple that automated equipment is now available to accomplish this crucial step. Adding a solvent. Once a dry residue of [18F]fluoride ion, together with the chosen counterion (either potassium/Kryptofix or a tetraalkylammonium salt), is obtained, it is necessary to dissolve the residue in an organic solvent appropriate for the subsequent chemical syntheses. In general, dipolar aprotic solvents are chosen, as many (but not necessarily all!) organic molecules used as substrates dissolve in them and they readily solublize the fluoride ion (particularly the K + /Kryptofix pair). Solvents such as dimethylsulfoxide and dimethylformamide also do not readily react with the fluoride ion; in addition to being a strong nucleophile, fluoride ion is also a strong base, and reactions performed in polar protic solvents (such a methanol) fail. The majority of [18F]fluorination reactions using fluoride ion are done in solvents such as dimethylsulfoxide, dimethylformamide or acetonitrile. Nucleophilic [18F]fluorination reactions are however not limited to these solvent choices, and have been performed in ethereal solvents such as tetrahydrofuran, and chlorinated solvents such as dichloromethane. The choice of solvent is more often predicated on the solubility of other reagents, the type of chemical reaction being performed, or simplification of the subsequent work-up and product isolation procedure. After many years of effort, these procedures have been adapted into automated apparatus which reliably produce reactive [18F]fluoride ion for radiopharmaceutical syntheses. Some of the concerns which troubled the field for many years have now effectively disappeared. Whereas specialized reaction vessels were employed for many early syntheses, such as glassy carbon or siliconized glass (Vacutainers), today reactions are successfully performed in simple borosilicate glassware. The uses of good commercial sources of [18O]water, and cyclotron targets made of such metals as titanium, tantalum, and silver, have removed the problems encountered earlier with unreactive [18F]fluoride ion due to competing anions or cations in the target water solution, which seemed to diminish the reactivity of resolublized [18F]fluoride ion. Finally, despite some earlier concerns that traces of fluorine-19 in reagents and solvents might effectively limit the achievable specific activities, high specific activity radiotracers (>2000 Ci/mmol) are now produced routinely at essentially all institutions. Aliphatic Nucleophilic Displacements The reaction of [18F]fluoride ion with a variety of leaving groups forms an excellent method for the synthesis of aliphatic carbon-fluorine bonds. Leaving groups range from simple halogens, to the common sulfonate

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esters (methanesulfonate, p-toluenesulfonate, trifluoromethanesulfonate), to cyclic structures such as epoxides or cyclic sulfates. Choice of leaving group may depend less on actual yield or rate of reaction with [18F]fluoride ion than on the availability or stability of precursors, the ease of subsequent separation of [18F]fluorinated product from precursors, reagents and solvents, or formation of potential side products. Trifluoromethanesulfonate esters, commonly called Inflates, are particularly reactive and provide excellent yields in nucleophilic [18F]fluorination reactions, such as in the synthesis of 2-deoxy-2-[18F]fiuoro-Dglucose. During the course of any nucleophilic substitution reaction, the formation of other products due to inadvertent reaction with other nucleophiles (purposefully added or not) should always be considered: such products either need to be separated from the final product or be so innocuous as to be allowed in the final radiopharmaceutical preparation. A second potential problem in nucleophilic aliphatic substitutions is elimination to form alkenes, a problem which has arisen in synthesis of some fluorinated cyclic structures. Aromatic Nucleophilic Substitution Fluorine-substituted aromatic rings make regular appearances in drug structures; fluorine is small and makes little impact on steric bulk, but the high electronegativity of the fluorine substituent can provide useful changes in the physiochemical and electronic characteristics of the aryl ring. Fluorination of aryl rings by nucleophilic displacements forms one example of significant differences between organic chemistry and radiochemistry: nucleophilic aromatic substitutions performed in the organic/medicinal chemistry laboratory often require high temperatures and long reaction times for completion (often at mediocre yields), but the reaction proceeds very rapidly, at moderate temperatures, and in high yields when done at the no-carrieradded level using [18F]fluoride ion. Nucleophilic aromatic substitution has become a method used widely in fluorine-18 chemistry, and there are some common characteristics of all applications, noted in the following sections. (a) As a substitution reaction, there is by definition a need for a leaving group on the aromatic ring. Nitro and trimethylammonium groups are the most widely utilized leaving groups in aromatic substitutions with [18F]fluoride ion. More recently newer groups, such as diphenyliodonium, have been proposed, and although they have some advantages in terms of not needing activating groups on the ring (see below) they have some specific activity limitations at the present. Simple isotopic substitution, [18F]fluoride for [19F]fluoride, can be a quick and effective method for synthesis of a new radiotracer, but the low specific activity achieved by these isotopic substitutions makes this unsuitable except where specific activity of the final product is of no concern. Direct comparisons of nitro and trimethylammonium groups have been made, but there has been no consistent conclusion as to which is more high yielding in [18F]fluorination reactions. There is, however, a considerable difference in the subsequent purification of the [18F]fluoroaromatics. Precursors bearing the nitro-substituted aryl rings are generally carried onward in any synthesis and the final product needs to be separated from the impurity; and unfortunately in many instances nitro- and fluoro-substituted arornatics have remarkably similar chromatographic properties. Although the nitro group could be reduced to an amine and that more easily separated from the fluoroaromatic, that extra step involves both complexity and additional time (loss of product by decay, even if quantitative yield). In contrast, the trimethylammonium group is permanently charged: the precursor is thus chromatographically very different from the

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fluoroaromatic, and can be easily separated, sometimes by relatively simple chromatographic methods. For that reason it is particularly appealing, although synthesis of the trimethylammonium group can be more difficult than the equivalent nitro-substituted precursor. (b) For nucleophilic substitution to proceed at anything resembling a reasonable rate, the aromatic rings need to be activated by the presence of one or more electron-withdrawing groups positioned ortho- or parato the leaving groups. A wide variety of substituents can function as electron withdrawing groups, including nitro, ketones, aldehydes, nitriles, esters, and amides. Studies utilizing carbon-13 NMR have shown a direct correlation between withdrawing power of a substituent and yields in nucleophilic aromatic substitutions with [18F]fluoride. The choice of activating group often depends on the structure of the desired final product, or the sequences of synthetic steps to follow incorporation of [18F]fluoride ion. For example, the activating group may form a part of the desired final product, or can be conveniently transformed into the needed structural component. Alternatively, activating groups can be transiently placed on the aryl ring and subsequently completely removed, a process that may be cumbersome but necessary for placement of [18F]fluoride at positions of a ring that cannot be activated towards substitution in the normal fashion. ELECTROPHILIC FLUORINATION A variety of electrophilic [18F]fluorination reagents have been developed and applied to synthesis of radiotracers. The original fluorinating agent was of course [18F]F2, but that can be a very highly reactive and destructive reagent. Numerous attempts have been made to "tame" the reactivity of fluorine gas by conversion to a second reagent. These include acetyl hypofluorite, xenon difluoride, diethylaminosulfur trifluoride, perchloryl fluoride (ClO3F), nitrosyl fluoride, N-fluoro-W-alkylsulfonamides, N-fluoro-2pyridone, N-fluoropyridinium triflates, N-fluoro-N-methyltrifluoromethane sulfonamide, N-fluoro-Wmethylnonafluorobutane sulfonamide, N-fluoro-A^-ethylperfluorooctane sulfonamide, and N-fluorobis(trifluoromethane sulfone)imide. Most of these new reagents have not been evaluated as to their scope and limitations for fluorine-18 labeling reactions. All of these are prepared from [18F]F2 produced in a carrier-added method, and thus all electrophilic fluorinations are necessarily carrier-added and result in final radiotracers with low to at best moderate specific activities. This has limited the usefulness of electrophilic fluorinations to the synthesis of radiopharmaceuticals for which there is not a need for high specific activity, and where the chemical species in question is not toxic. A second drawback of electrophilic fluorination is that the maximum achievable radiochemical yield is only 50%; for reactions with [18F]F2 only one of the two fluorine atoms is incorporated into the product, and reagents such as acetyl [18F]hypofluorite are synthesized from [18F]F2 and half the radioactive atoms are lost in the preparation of the labeled fluorinating reagent. Despite the drawbacks such as low specific activity, reduced radiochemical yields, and poor regioselectivity (discussed below), electrophilic fluorination has been the method initially used to prepare radiopharmaceuticals which subsequently proved important, including [18F]FDG and [l8F]fluoroDOPA. Only much later were higher yield, regioselective syntheses developed for these radiotracers, using high specific activity nucleophilic [18F]fluoride ion.

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Aliphatic electrophilic fluorination The addition of elemental [18F]fluorine gas to an alkene was the method used for the original synthesis of [!8F|FDG, but that synthesis was replaced by syntheses using the milder reagent acetyl hypofluorite (and later by a nucleophilic fluorination route). Today, fluorinations of alkenes are rarely used for the synthesis of fluorine-18 radiotracers, however there are places where this synthetic method does have applications. One is the preparation of fluorine-18 labeled perfluoroalkyl groups, such as the hypoxic sensitizer EF5, where application of electrophilic fluorination may work but nucleophilic routes using [18F]fluoride ion are unlikely to succeed. Aromatic electrophilic fluorination Electrophilic reagents such as [18F]F2 or acetyl [18F]hypofluorite are very reactive, and addition to solutions of aromatic compounds results in addition-elimination reactions to yield aryl [18F]fluorides. Such reactions are however non-regioselective, and more often than not result in a mixture of [18F]fluorinated products. This reduces again the maximum yield of any single desired fluorinated product, but can be used to advantage in the development phase of new radiopharmaceuticals, as several positional isomers of [18F]fluoroaromatics can be prepared and evaluated in biological tests using a single [18F]fluorination reaction. Regioselective fluorinations can be achieved using [18F]fluorodemetallation reactions. In these syntheses, fluorine replaces a metal substituent such as a trialkyltin group or mercuryl halide. Similar reactions are successful with silicon or germanium, but the highest yields are often produced with the tin reagents. FLUORINE-18 LABELED RADIOPHARMACEUTICALS: A GROWING LIST Ten years ago there were already several hundred compounds which had been labeled with fluorine-18 (Kilbourn, 1990). In the intervening decade there has been a veritable explosion in the fluorine-18 literature, both in the sheer number of new radiotracers published and in the number of these reaching at least preliminary human evaluations. The list has simply grown far beyond the scope of this review to list and reference. The remainder of this chapter is devoted to short discussions of a few significant fluorine-18 labeled radiotracers and their various preparations. This discussion has been limited, mostly, to radiopharmaceuticals which have reached clinical application using Positron Emission Tomography and to some closely related compounds which, in the authors' opinion, may do so in the near future.

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Table 2. Fluorine-18 labeled radiopharmaceuticals in clinical use. Compound 18

6-[ F]FluoroDOPA 18

6-[

F]Fluoro-m-tyrosine

2-[18F]Fluorophenylalanine 18

2- [ F]Fluoro-4-boronophenylalanine [18F]Fluoroethyltyrosine 18

[ F]Fluoro-a-methyltyrosine 18

6-[ F]Fluorodopamine (-)-6-[18F]Fluoronorepinephrine [18F]Setoperone [18F]Altanserin 18

[ F]N-Methylspiperone [18F]Fluoroethylspiperone [l8F]Fluoropropylspiperone [l8F]Fluoromisonidazole

Common synthesis Electrophilic Fluoro-demetallation nucleophilic aromatic substitution of nitro fluorodestannylation

Clinical utility dopamine metabol-

ism

electrophilic fluorination with [18F]AcOF electrophilic fluorination with [18F]AcOF 0-alkylation with [l8F]fluoroethyl tosylate electrophilic fluorination with [18F]AcOF nucleophilic aromatic substitution of 6-nitropiperonal nucleophilic aromatic substitution nucleophilic aromatic substitution of nitro nucleophilic aromatic substitution of nitro nucleophilic aromatic substitution of nitro N-alkylation with [ l8 F]fluoroethyl tosylate N-alkylation with 3[18F]fluoroiodopropan

References (Adam & Jivan, 1988; Namavari et al., 1992) (Ding ei al. 1990; Lemairee/a/., 1994) (Namavari et al., 1993)

Dopamine metabolism neutral amino acid transport neutron capture therapy titration tumor imaging

(Wester etal., 1999b)

tumor imaging

(Tomiyoshiera/.. 1997)

cardiac sympathetic innervation

(Ding etal., 1991b)

cardiac sympathetic innervation 5HT2A receptors

(Ding et at., 199 la) (Crouzdetai, 1988)

5HT2A receptors

(Lemaireera/., 1991b)

D2 receptors

(Hamacherera/.. 1986)

D2 receptors

(Block etal.. 1986)

D2 receptors

(Shiueefa/.. 1987)

hypoxia imaging

(Griersonetal., 1989)

tumor imaging

(Fowlers a!.. 1973)

(Coenene/a/.. 1988; Murakami e/ al., 1988b) (Ishiwata^a/., 1991b)

e N-alkylation using [ l8 F]epifluorohydrin direct electrophilic fluorination with [18F]F2 nucleophilic displacement of aliphatic cyclic sulfone nucleophilic displacement of mesylate electrophilic 18F-for-l9F exchange electrophilic l8F-for-19F exchange

estrogen receptor positive tumors

(Lim etal., 1996: Romer etal., 1996)

Antibiotic pharmacokinetics Antibiotic pharmacokinetics Antibiotic pharmacokinetics

(Uvmietal., 1993)

[18F]Fluconazole

nucleophilic aromatic Schiemann reaction

Antibiotic pharmaco-kinetics

(Livinietal., 1992)

[18F]CFT

fluorodestannylation with high spec. act. [18F]AcOF

dopamine transport

(Haaparantaera/., 1996)

[18F]FP-CIT

N-alkylation with [18F]fluorobromoprop

dopamine transport

(Lundkvistera/.. 1997)

18

5-[ F]Fluorouracil 16a-[18F]Fluoroestradiol [18F]Fleroxacin [18F]Trovafloxacin 18

[ F]Lomefloxacin

(Babichera/., 1996) (Tewsonetal., 1996)

ane

A list of fluorine radiotracers reported for human use is given in Table 2 along with the "most used" method of synthesis. This is by no means a complete listing but hopefully represents an adequate cross-section of both radiochemical methods and clinical uses.

Using computerized database searches even syntheses

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203

reported in the most obscure journals can be located and eventually obtained, either in paper form or increasingly in electronic journal format. Readers interested in finding examples of specific radiochemical reactions, or certain types of compounds or drugs, are encouraged to employ such methods to find and read the burgeoning chemical and biological literature. A separate chapter in this volume has been devoted entirely to a detailed discussion of 2-deoxy-2-[l8F]fluoroD-glucose ([18F]FDG) and its importance to the field of Nuclear Medicine. As such, [18F]FDG has been excluded from Table 2 and its chemistry will not be discussed here. Among those radiotracers listed in Table 2, L-3,4-dihydroxy-6-[18F]fluorophenylalanine ([18F]FDOPA) remains far and away the most often cited fluorinated radiopharmaceutical over the past decade, other than [18F]FDG. This is followed, and not closely, by [l8F]setoperone and [18F]altanserin for the serotonin 5HT2A receptors and the 18F-labeled spiperone derivatives for dopamine D2 receptors. [18F]Fluoroestradiol ([I8F]FES) and 5-[!8F]fluorouracil (5-[18F]FU) seem finally to be being used more in the past 3 or 4 years whereas use of [18F]fluoromethane seems to have dwindled, [18F]fluoromisonidazole, [18F]setoperone and [18F]altanserin have been the subject of comprehensive reviews (Crouzel et al., 1992; Stocklin, 1995; Grierson & Patt, 1999) since which no further significant improvements have been reported, and thus none of these latter radiotracers will be discussed further here. AROMATIC AMINO ACIDS All three naturally occurring aromatic amino acids, phenylalanine, tryptophan and tyrosine, have been labeled with fiuorine-18 through electrophilic substitution methods analogous to those discussed later for L3,4-dihydroxy-6-[18F]fluorophenylalanine ([18F]FDOPA) (Hoyte et al., 1971; Atkins et al., 1972; Goulding & Palmer, 1972; Goulding & Gunasekera, 1975; Goulding & Clark, 1979; Coenen et al, 1986; Coenen et al., 1988; Murakami et al., 1988a; Murakami et al., 1988b). Very little has been published concerning [!8F]fluorotryptophan beyond early reports of its successful synthesis using electrophilic substitution (Atkins et al., 1972; Goulding & Clark, 1979). Similarly, only a few publications have appeared using 3[!8F]fluorotyrosine ([18F]F-Tyr) for imaging. It was initially reported that this radiotracer could be used as an index of protein synthesis for oncology (Coenen et al., 1989; Vaalburg et al., 1992). Later studies showed that, although [18F]F-Tyr accumulated in tumors, the actual process being measured was neutral amino acid transport rather than protein synthesis (Wienhard et al., 1991; Ishiwata et al., 1993b). Two tyrosine derivatives, 0-[18F]fluoroethyl-L-tyrosine and 3-[18F]fluoro-a-methyltyrosine have recently been studied for imaging brain tumors. O-[18F]fluoroethyl-L-tyrosine ([18F]FET) is prepared in two steps starting with nucleophilic [18F]fluorination of ethylene glycol-l,2-ditosylate using K[l8F]F/Kryptofix followed by reaction of the intermediate 2-[18F]fluoroethyl tosylate with the di-potassium salt of L-tyrosine (Wester et al., 1999b). This procedure provided [18F]FET an overall decay-corrected radiochemical yield of 40%, in a total synthesis time of 60 min. Imaging results for [!8F]FET in a series of patients with suspected primary or recurrent intracerebral tumors were comparable to [HC]methionine (Weber et al., 2000). Similarly, 3-[18F]fluoro-a-methyltyrosine (18F-AMT) exhibited tumor-to-background ratios significantly higher than [18F]FDG in brain tumor patients (Inoue et al., 1999). The synthesis of 18F-AMT was accomplished via direct electrophilic fluorination of a-methyltyrosine with acetyl [18F]hypofluorite

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(Tomiyoshi et al, 1997). This reaction gave a 15:85 mixture of the 2- and 3-[!8F]fluoro isomers which was used without separation. The utility of 2-[18F]fluorophenylalanine to assess neutral amino acid transport both in normal human brain and tumor tissues has been investigated (Ito et al., 1995; Ogawa et al., 1996; Mineura et al., 1997a: Mineura et al., 1997b). Early studies found that use of [I8F]F2 as the fluorinating reagent gave all three possible [18F]fluorophenylalanine isomers (Coenen et al., 1986; Murakami et al., 1988a). However, direct fluorination of phenylalanine with acetyl [18F]hypofluorite provides only 2-[18F]fluorophenylalanine (Coenen et al., 1988; Murakami et al., 1988b). This strategy was also put to use in the synthesis of 4-borono-2[18F]fluoro-D,L-phenylalanine, a PET radiotracer used for the development of neutron capture therapies for oncology (Ishiwata et al., 1991a; Ishiwata et al., 1991b; Ishiwata et al., 1992; Imahori et al., 1998a: Imahori etal, 1998b; Imahori et al., 1998c). 6-r'8F1FluoroDOPA (T18F1FDOPA). The chemistry and utility of L-3,4-dihydroxy-6-[18F]fluorophenylalanine (6-[18F]fluoro-L-DOPA, [18F]FDOPA) has been discussed, to varying degrees, in numerous reviews owing to its widespread use and to continued discussions regarding both methods of production and interpretation of PET imaging data from this radiopharmaceutical (Fowler & Wolf, 1990; Fowler, 1993; Halpem, 1995; Ding & Fowler, 1996; Ruth & Adam, 1996; Ding, 2000). Two particularly exhaustive radiochemistry reviews report the groundbreaking work in the synthesis of [18F]FDOPA (Luxen et al., 1992; Stocklin, 1995). Preparation of [18F]FDOPA can be accomplished by either electrophilic or nucleophilic procedures, both of which have certain limitations. Regioselective electrophilic fluorodemetallation of either the mercuryl (Adam & Jivan, 1988; Luxen et al., 1990) or trimethylstannyl (Namavari et al., 1992) precursor are rapid, simple (and therefore amenable to automation) and provide enantiomerically pure [I8F]FDOPA in up to 25% radiochemical yield (decaycorrected). In fact, fully automated synthesis modules for both the fluorodemercuration (Chaly et al., 1994; Szajek et al., 1998) and fluorodestannylation (deVries et al., 1999) reactions have recently been reported. Also with an eye toward automation, the preparation of a simplified, fully tert-butoxycarbonyl-protected precursor for fluorodestannylation has been described (Dolle et al., 1998). However, as in all electrophilic fluorination procedures, preparation by this method of sufficient amounts of [18F]FDOPA for multiple patients is limited by the low efficiency of [18F]F2 production and the maximum theoretical yield of 50%. The low specific activity achieved with this carrier-added synthesis does not pose a problem with the utility of this tracer. With the use of mercuryl or trimethylstannyl precursors, it is necessary to perform analyses to verify complete removal of these potentially toxic metals from the final dose formulation. For this reason, recent efforts have focused on attempts to develop regioselective syntheses that do not utilize metallic precursors (Ishiwata et al., 1993a) or that have the metallated precursor bound to a polystyrene resin (Kawai et al, 1995; Szajek et al., 1998). Nucleophilic methods using no-carrier-added [18F]fluoride ion (Ding et al., 1990; Lemaire et al., 199la; Lemaire et al., 1992), have the potential to provide a higher absolute yield and higher specific activity. These are more complex multi-step syntheses, typically taking 2-3 hours, and thus are more difficult to automate; nevertheless, a semi-automated nucleophilic synthesis of [18F]FDOPA has been accomplished

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(Reddy et al., 1993). The use of chiral auxiliaries (Lemaire et al., 1993; Horti et al., 1994; Lemaire et al., 1994) or a chiral catalytic phase-transfer procedure (Lemaire et al., 1999) can provide [18F]FDOPA in 90– 98% enantiomeric excess of L-[18F]FDOPA, thus eliminating the necessity for HPLC resolution of the racemic mixture. The phase-transfer procedure also had the advantage of a somewhat shorter (90 min) synthesis time, although it was also the least enantioselective (90% e.e.) of the procedures reported. An enzymatic synthesis of [18F]FDOPA from 4-[' F]fluorocatechol (prepared via nucleophilic aromatic substitution with no-carrier-added [18F]fluoride ion) through the action of p-tyrosinase has the advantage of producing enantiomerically pure [18F]FDOPA from commercially available precursors (Kaneko et al., 1999). Synthesis times are the usual 2.5 hours and the product is isolated in a modest 2% (decay-corrected) radiochemical yield. Alternatively, a chiral precursor for [18F]FDOPA, (25,55)-5-(3-formyl-6-iodo-4methoxybenzyl)-l-f-butoxycarbonyl-2-r-butyl-3-methyl-4-imidazolidinone, has recently been described (Kuroda et al., 2000). Presumably nucleophilic fluorination followed by Baeyer-Villiger oxidation and deprotection would provide enantiomerically pure [18F]FDOPA, but no results of such radiochemical syntheses have been provided. In order to prepare sufficient amounts of [18F]FDOPA for use with multiple patients, or for transport and distribution to sites distant from the site of synthesis, will require either a streamlined nucleophilic synthesis or improved yields from the electrophilic production method. Perhaps the new preparations of [18F]F2 in higher yields and specific activity from no-carrier-added [18F]fluoride ion (Bergman & Solin, 1997), and subsequent conversion to acetyl hypofluorite using standard methods (Fowler et al., 1982), will help overcome these challenges through a hybrid synthesis. Fluoro-L-m-tyrosine All three isomers of [18F]fluoro-meta-tyrosine ([18F]FmT) have been evaluated, in both normal and MPTPlesioned primates, as PET radiotracers to replace [18F]FDOPA for imaging dopamine metabolism (Melega et al., 1989; Hayase et al., 1994; Hayase et al., 1995; Barrio et al., 1996; DeJesus et al., 1997; Jordan et al., 1997; Brown et al., 1999; Doudet et al., 1999). Although initial syntheses using direct electrophilic fluorination of m-tyrosine with acetyl [18F]hypofluorite gave a mixture of 2-, 4- and 6-[18F]FmT (DeJesus et al., 1990), a regiospecific fluorodestannylation procedure has been reported to give either 4- or 6-[18F]FmT (Namavari et al., 1993). Synthesis of 4-[18F]FmT has also been accomplished using fluorodemercuration (Perlmutter et al., 1990). Similar to [18F]FDOPA, both 4- and 6-[18F]FmT exhibit an in vivo distribution in monkey brain that correlates well with known concentrations of aromatic L-amino acid decarboxylase (Hayase et al., 1994; Barrio et al., 1996; Brown et al., 1999). In contrast to [18F]FDOPA, these radiotracers are not substrates for catechol O-methyl transferase and thus do not form the 3-O-methylated metabolite that has so hindered the quantitative analysis of FDOPA (Melega et al., 1989; Nahmias et al., 1995; Jordan et al., 1997; Jordan et al., 1998). Their distribution is not restricted to dopaminergic areas and appears inadequate to estimate dopamine turnover rates (Brown et al., 1999; Doudet et al., 1999). Evaluation of 6-[18F]FmT in human subjects is very preliminary (Nahmias et al., 1995; Wahl et al., 1999) and it remains to be seen whether this radiotracer represents a viable alternative to [18F]FDOPA.

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PHENETHYLAMINES [18F1Fluorodopamine and [18F]fluoronorepinephrine The norepinephrine analogs (-)-6-[18F]fluoronorepinephrine (6-[18F]FNE), and 6-[18F]fluorodopamine ([18F]FDA) have been developed as radiotracers for peripheral sympathetic innervation. The early radiochemistry and relative advantages of these two radiotracers for cardiac imaging have been discussed elsewhere (Fowler, 1993; Stocklin, 1995). The development of radiolabeling procedures has closely paralleled that of [18F]FDOPA with both electrophilic and nucleophilic syntheses reported. However, unlike [18F]FDOPA, [18F]FDA and [18F]FNE can cause hemodynamic effects which limits the injectable dose of these radiotracers if prepared in low specific activities. Several recent electrophilic labeling procedures, using either direct fluorination (Chirakal et al., 1996), fluorodemercuration (Chaly et al., 1993) or fluorodestannylation (Culbert et al., 1995; Namavari et al., 1995), have been reported. However, none of these procedures offers any substantial improvement of either radiochemical yield or specific activity. The nucleophilic fluorination of [18F]FDA uses commercially available 6-nitropiperonal in a procedure similar to that first reported for [18F]FDOPA (Ding et al., 1991b). Although radiochemical yield and total synthesis time for this procedure (20%, decay corrected to end-of bombardment (EOB), in 105 min) are similar to the electrophilic methods, the specific activity of the[18F]FDA prepared is > 2000 Ci/mmol as compared to 10 Ci/mmol for direct electrophilic fluorination (Chirakal et al., 1996). A similar nucleophilic procedure for the preparation of (-)-6-[18F]fluoronorepinephrine (6-[18F]FNE) also provides high specific activity radiotracer in modest 6% (EOB) radiochemical yield (Ding et al., 199la). This procedure has the disadvantage of producing a racemic mixture of (+)- and (-)-6-[18F]FNE which must be resolved chromatographically. A stereospecific enzymatic synthesis of (-)-6-[18F]FNE, using dopamine (Bhydroxylase catalyzed oxidation of [18F]FDA, has been reported (Lui et al., 1998). However, this enzymatic step requires 70 minutes in addition to the 1-2 hour synthesis of [18F]FDA, and thus significantly reduces the isolated yield. The stereoselective synthesis of (-)-6-[18F]FNE will likely benefit from chiral auxiliary methodology similar to that used for [18F]FDOPA. [18F]Fluorometaraminol Metaraminol is another norepinephrine (NE) analog that, like NE, is transported into sympathetic neurons and stored in vesicles, but unlike NE, is not a substrate for the major metabolic enzyme monoamine oxidase (MAO) (Fowler, 1993; Stocklin, 1995). N-[11C]Methyl metaraminol, more commonly known as m[11C]hydroxyephedrine ([11C]HED), has proven to be an important radiopharmaceutical for cardiac imaging (Rosenspire et al., 1990). 6-[18F]Fluorometaraminol (6-[18F]FMR) was originally prepared by direct electrophilic fluorination of protected metaraminol using acetyl [l8F]hypofluorite (Mislankar et al., 1988). Studies in canine heart showed 6-[18F]FMR to be a promising radiotracer for cardiac imaging. However, as was the case with [18F]FDA and [18F]FNE, use of 6-[18F]FMR in humans is limited by specific activity considerations. Recently two no-carrier-added syntheses of the para-fluorinated analog, 4[18F]fluorometaraminol, using nucleophilic aromatic substitution of a trimethylammonium leaving group, have been reported. Both make use of the benzylic oxygen substituent to activate the substitution reaction by

CHEMISTRY OF FLUORINE-18 RADIOPHARMACEUTICALS starting with the p-trimethylammonium

triflate derivative of either the fully benzyl-protected

20? a-

aminopropiophenone (Ermert, 1998) or 0-benzylbenzaldehyde (Langer et al., 1999). Radiofluorination followed by reduction/deprotection of the former precursor with borane-tetrahydrofuran complex gave predominantly erythro-4-[18F]fluorometaraminol (4:1 erythro vs. threo). Nitroalkylation of the benzaldehyde precursor, followed again by reduction/deprotection by catalytic hydrogenation, gave a racemic mixture which was resolved by chiral separation on HPLC to give the active lR,2S-stereoisomer. The decaycorrected radiochemical yield in both cases was approximately 20% and specific activity was in excess of 2000 Ci/mmol. As no significant pharmacological differences were observed between the o- and p-fluoro compounds in initial animal studies (Langer et al., 2000), these high specific activity preparations of 4[18F]FMR should finally allow evaluation of this radiotracer in humans. STEROIDS 16-a-[18F]Fluoroestradiol Despite a large medicinal and radiochemistry effort over the past two decades (Palmer & Widdowson, 1979; Pomper et al., 1990; VanBrocklin et al., 1992; VanBrocklin et al., 1993a; VanBrocklin et al., 1994; Hosteller et al., 1999), 16a-[18F]fluoroestradiol-17p ([18F]FES) remains the only clinically relevant PET radiotracer for imaging estrogen receptor-positive breast tumors. The design and development of FES has been reviewed elsewhere (Katzenellenbogen, 1995; Katzenellenbogen et al., 1997). The first synthesis of [18F]FES, published over 15 years ago (Kiesewetter et al., 1984a; Kiesewetter et al., 1984b), utilized nucleophilic displacement of the aliphatic triflate of 3,16B-bis(trifluoromethanesulfonyloxy)estrone using tetrabutylammonium [18F]fluoride, followed by lithium aluminum hydride reduction of the fluoro-ketone intermediate. HPLC purification provided a 30% radiochemical yield of high specific activity [18F]FES in an overall synthesis time of 90 minutes (Kiesewetter et al., 1984b; Brodack et al., 1986). An improved one-pot synthesis of [18F]FES was originally published in abstract form by Lira et al. (1994), and later elaborated in simultaneous full publications by two groups (Lim et al., 1996; Romer et al., 1996). This method utilizes the cyclic sulfone of 3-methoxymethyl-16p,17p-epiestrol as a radiolabeling precursor. Due to steric considerations, nucleophilic attack by [l8F]fluoride ion, using either tetramethylammonium [18F]fluoride or [18F]KF/Kryptofix[2.2.2], occurred exclusively at the 16a position. Simultaneous hydrolysis of both the ring-opened sulfate ester and the 3-methoxymethyl protecting group by heating in dilute HC1, followed by HPLC purification, provided [18F]FES in up to 70% decay-corrected radiochemical yield with a synthesis time of only 70 minutes. This one-pot method has recently been adapted for automation and a synthesis module is now commercially available (Romer et al., 1999).

[18F]Fluoromoxestrol A number of other estrogen and progestin analogs have been labeled with fluorine-18 using nucleophilic displacement of the alkyl triflates, and two of these have been tested in humans. 16p-[l8F]Fluoromoxestrol ([18F]FMOX) (VanBrocklin et al., 1992; VanBrocklin et al., 1993b) is an estrogen designed to circumvent the rapid in vivo metabolism which complicates quantitative analysis of [18F]FES images (Katzenellenbogen,

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1995; Mankoff et al., 1997). Rodent data looked promising, with [18F]FMOX exhibiting slower in vivo metabolism and better tissue localization than [18F]FES. However, in human trials no tumor localization was seen, likely due to the presence of sex hormone-binding globulin which protects [18F]FES, but not [18F]FMOX, from metabolism in humans (Jonson et al., 1999). [18F]Fluoro-16a-ethyl-19-norprogesterone The progesterone receptor ligand 21-[18F]fluoro-16a-ethyl-19-norprogesterone ([18F]FENP) was synthesized in a one-step procedure from the corresponding 21-triflate ester and tetrabutylammonium [18F]fluoride (Pomper et al., 1988). Like [18F]FMOX, rodent studies with [18F]FENP were very promising but the radiotracer was not useful in human subjects due to rapid metabolism (Verhagen et al., 1994). However, sterically hindered analogs which may show improved metabolic profiles are under development (Buckman et al., 1995). BUTYROPHENONE NEUROLEPTICS Spiperone derivatives The butyrophenone neuroleptics are a class of dopamine D2 receptor antagonists which include four fluorine18-labeled radiotracers currently in human use, [18F]spiperone, [l8F]N-methylspiperone ([18F]NMS), 3-(2[I8F]fluoroethyl)spiperone ([18F]FESP) and N-(3-[18F]fluoropropyl)spiperone ([18F]FPSP) (Paans et al., 1996; Volkow et al., 1996; MacFarlane et al., 1997; Perlmutter et al., 1998; Stocklin, 1998). Although the N[11C]methylspiperone appears quite often in the recent neuroimaging literature, the publications concerning the corresponding fluorine-18 labeled neuroleptics have dwindled in the past few years. In fact, the most recent publications on [18F]FESP are not in neuroscience at all, but rather describe the utility of this radiotracer in tumor imaging (Lucignani et al., 1997) and as a reporter for gene therapy (MacLaren et al., 1999). The radiochemical syntheses of the fluorine-18 labeled butyrophenones have changed little since the comprehensive reviews by Kilbourn (1990), Maziere et al. (1992) and Stocklin (1995). Recent work has focused on optimizing the HPLC purification of radiotracers (Hashizume et al., 1995; Hashizume et al., 1997) and use of a trimethylanilinium triflate precursor for nucleophilic fluorination of [18F]spiperone (Banks et al., 1994) in place of the original nitrated precursor, cyclopropyl p-nitrophenyl ketone. A procedure for the synthesis of [18F]FPSP using 3-bromopropyl triflate, rather than the 1,3-dihalopropanes normally used, was also reported. The intention was to simplify purification and provide improved specific activity, but the total synthesis time for this procedure was longer than that reported using 1,3-diiodopropane (Shiue et al., 1987) and no quantitative specific activity results were provided.

N-methylbenperidol Another butyrophenone neuroleptic which shows promise as a PET radiotracer, but for which no human data is yet available, is [l8F](N-methyl)benperidol ([18F]NMB) (Moerlein et al., 1992). Unlike the spiperone derivatives, [18F]NMB is selective for dopamine D2 binding sites versus D1 and 5HT2 receptors (Moerlein et al., 1997). N-methylbenperidol was originally labeled with carbon-11 (Suehiro et al., 1990). but was not

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pursued, presumably due to the slow kinetics of this series of radiotracers (Moerlein et al., 1997). Both [18F]benperidol (Arnett et al., 1985) and 3-(2'-[18F]fluoroethyI)benperidol (Moerlein & Perlmutter, 1992) have also been prepared previously but likewise have not been pursued in the recent literature. The preparation of [18F]NMB is analogous to the three-step synthesis of [18F]N-methylspiperone, using cyclopropyl p-nitrophenyl ketone as the precursor and providing [18F]NMB in a decay-corrected radiochemical yield of 5-10% with a total synthesis time of 100 min for the three steps (Moerlein et al., 1992). The specific activity of the product is reported as >3000 Ci/mmol. TROPANES The radiochemistry literature has been dominated over the past several years by synthetic approaches to labeled cocaine analogs designed to image neuronal membrane monoamine transporters (Carroll et al., 1995). In particular, a number of 2p-carbomethoxy-3p-phenyltropane derivatives have been labeled with either carbon-11, fluorine-18 or iodine-123 and their utility for imaging the dopamine transporter evaluated. Two of the fluorine-18 labeled radiotracers, 2p-carbomethoxy-3p-(4-[18F]fluorophenyl)tropane ([18F]CFT or [18F]WIN 35,428) and N-[18F]fluoropropyl-2p-carbomethoxy-3p-(4-iodophenyl)nortropane ([18F]FP-CIT, [I8FJCIT-FP or [18F]FPCIT) have undergone clinical evaluations in normal humans (Chaly et al., 1996; Laakso et al., 1998) and patients with Parkinson's disease (PD) (Kazumata et al., 1998; Rinne et al., 1999). Such radioligands exhibited specific accumulation in dopamine-rich brain regions and were able to detect striatal dopaminergic deficits, which proved to be relatively large even in early PD and illustrated the potential utility of such radiotracers for in vivo imaging. The synthesis of [18F]CFT was accomplished by electrophilic aromatic substitution of the stannylated precursor using higher specific activity [18F]acetyl hypofluorite (Haaparanta et al., 1996). The overall radiochemical yield of this 65-75 minute synthesis was low (1-2% at end of synthesis (EOS)). However, by utilizing high specific activity [18F]F2 (Bergman & Solin, 1997), which was subsequently converted to [18F]acetyl hypofluorite by conventional methods (Fowler et al., 1982), a [18F]CFT specific activity of 300435 Ci/mmol (EOS) was achieved. The modest success of this method lends encouragement to its possible use for other radiotracers such as [18F]FDOPA. N-Fluoropropyl-2p-carbomethoxy-3p-(4-iodophenyl)nortropane (FP-CIT) has been labeled with carbon-11 (Lundkvist et al., 1995), fluorine-18 (Chaly et al., 1996; Lundkvist et al., 1997), and iodine-123 (Neumeyer et al., 1991). The [18F]fluoropropyl analog was originally prepared by a standard nucleophilic fluorination of the methanesulfonate using K[18F]F/Kryptofix (Chaly et al., 1996). As with the preparation of [18F]CFT, radiochemical yields were only 1 -2% (EOS) and the total synthesis time was 80 min. No mention was made of specific activity for this synthesis. An alternative two-step route utilizes [18F]fluorobromopropane, synthesized from 1,3-dibromopropane and K[18F]F/Kryptofix (Lundkvist et al., 1997). Subsequent alkylation of nor-p-CIT provided a slightly improved 2-4% radiochemical yield and specific activity of 1500 Ci/mmol (EOS). The total synthesis time for this two-step procedure was still only 90 min. A number of other fluorine-18 labeled tropanes have been synthesized but evaluation has not yet reached the stage of human trials. Among these are N-[18F]fluoroethyl-2p-carbomethoxy-3p-(4-chlorophenyl)nortropane

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([18F]FECNT) (Goodmans al., 2000), N-4-[18F]fluorophenyl)-methyl-2p-carbomethoxy-3p-(418 chlorophenyl)nortropane ([ F]FCT) (Mach et al., 2000), 2p-carbomethoxy-3p-(418 [ F]fluoromethyl)phenyltropane (p-FWIN) (Petric et al., 1999; Stout et al., 1999) and 2p-carbo-l[18F]fluoro-2-propoxy-3p-(4-chlorophenyl)tropane ([18F]FIPCT) (Xing et al., 2000). The synthesis of all of these compounds except [18F]FIPCT showed large improvements in radiochemical yield (~20-25% EOS) over current radiotracers. However, the utility of these compounds for PET imaging in humans remains to be determined. ANTIBIOTICS Fluoroquinolones The fluoroquinolone class of antibiotics are bacteria DNA gyrase inhibitors exhibiting an extremely broad spectrum of activity against both gram-positive and gram-negative organisms (Fischman et al., 1992). Fluorine-18-labeled radiotracers based on these compounds have been used almost exclusively for determination of in vivo drug pharmacokinetics (Fischman et al., 1993b; Fischman et al., 1996; Tewson et al., 1996; Fischman et al., 1998). In these investigations the radiotracers are co-injected with pharmacological doses of unlabeled antibiotic and the distribution of drug in both infected and normal tissues is determined. Researchers are then able to determine whether sufficient accumulation in infected tissues is achieved, when peak concentrations are reached and how long pharmacologically relevant concentrations are maintained. Such use of in vivo imaging as a tool for pharmaceutical development is highly powerful yet largely unexploited. Three fluoroquinolones have been labeled with fluorine-18 for use in human volunteers. The first of these was [18F]fleroxacin, which was synthesized in a two-step procedure from the corresponding methanesulfonate, with the carboxylic acid moiety protected as the ethyl ester (Fischman et al., 1992; Livini et al., 1993). Nucleophilic displacement of the methanesulfonate with K[18F]F/Kryptofix, followed by alkaline hydrolysis of the ethyl ester provided [18F]fleroxacin in a modest uncorrected 5-8% radiochemical yield (Livini et al., 1993). Total synthesis time was 90 minutes. As specific activity is irrelevant in these carrier-added pharmacokinetic assays, the other two fluoroquinolone radiotracers, [18F]lomefloxacin (Tewson et al., 1996) and [18F]trovafloxacin (CP 99,219) (Babich et al., 1996) were synthesized using a [l8F]fluorine-for-[19F]fluorine exchange reaction on the aromatic fluorine substituents. Lomefloxacin is a longer acting antibiotic than fleroxacin, and trovafloxacin is effective against fleroxacin-resistant bacterial strains. Both were prepared in 15-30% radiochemical yield (EOS) from K[18F]F/Kryptofix by heating at 160 °C for 60 minutes for [l8F]lomefloxacin and 30 minutes for [l8F]trovafloxacin. This lead to total synthesis times of 90 and 70 minutes, respectively. In both cases, higher temperatures or extended reaction times led to significant decomposition of both starting material and radiotracer. The purification of [l8F]lomefloxacin was interesting in that it was achieved by mixing the crude dimethylsulfoxide reaction solution with 600 mg of unlabeled lomefloxacin and recrystallizing this bulk mixture from hot ethanolic HC1. Recovery of the amine hydrochloride after cooling for 20 minutes and

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filtration was 75-80% which represented 35-40 mCi of radiotracer (Tewson et al., 1996). This procedure was made radiochemically feasible by a unique pairing of nucleophilic [18F]fluorination with an assay that doesnt require high specific activity radiotracer. [18F]Trovafloxacin was purified by a more traditional HPLC method (Babich et al., 1996). Fluconazole Fluconazole, is an antifungal agent structurally unrelated to the fluoroquinolone antibiotics but which has been labeled with fluorine-18 and utilized in similar low specific activity pharmacokinetic investigations (Fischman et al., 1991; Fischman et al., 1993a). Preparation of [18F]fluconazole makes use of an interesting nucleophilic aromatic fluorine-for-amine substitution using a modified Schiemann reaction (Livini et al., 1992). In this procedure the aniline starting material, K[i8F]F/HBF4 and sodium nitrite were mixed in aqueous solution and the water evaporated under nitrogen at 55 "C. Heating the residue at 130-135 °C for 30 minutes provided, after NH2-Sep-Pak purification, a modest 1-2% radiochemical yield (EOS) of [!8F]fluconazole in >95% radiochemical purity. Due to this low reaction yield, and synthesis times are around 2 hours, it was necessary to start with >500 mCi of [18F]fluoride ion in order to produce sufficient radiotracer for human use. However, this in situ diazatization procedure might be more effective when applied to the electron-deficient systems typically used for nucleophilic aromatic [l8F]fluorinations.

5-FLUOROURACIL The commonly used cancer chemotherapeutic, 5-fluorouracil (5-FU), has a long history of use for diagnostic imaging in oncology. The radiolabeling of 5-FU with fluorine-18, using a recoil labeling procedure, was reported in 1972 (Lebowitz et al., 1972). In this procedure, the radiolabeling precursor, in this case uracil, is coated directly on the walls of a neon-20 gas target and the target irradiated with a deuteron beam accelerated in a Van de Graaff generator. This technique worked but generated only a 1% yield of 5-[18F]FU. Early the next year, Fowler et al. reported a more traditional electrophilic synthesis of 5-[18FjFU, using uracil and [18F]F2 in trifluoroacetic acid (Fowler et al., 1973). This provided a much improved 17% radiochemical yield (EOB) of low specific activity (0.2 Ci/mmol) 5-[18F]FU in a total synthesis time of only 35 minutes. In the nearly three decades since these original publications, a number of modified electrophilic syntheses of 5-[18F]FU, using either [18F]F2 (Vine et al., 1979; Diksic & DiRaddo, 1984; Ishiwata et al., 1984; Oberdorfer et al,, 1989) or acetyl [18F]hypofluorite (Diksic et al., 1984; Visser et al., 1989), have been reported. These efforts have combined to provide improvements in radiochemical yield (~30% EOB) and chemical and radiochemical purity, but synthesis times are much longer, due to more complex purification procedures, and specific activities are still in the range of 1 Ci/mmol. Fortunately, as with [18F]FDOPA, specific activity doesnt seem to be an issue with respect to 5-[l8F]FU imaging efficacy.

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OTHER SMALL MOLECULE RADIOTRACERS IN DEVELOPMENT Fallypride Fallypride is a selective antagonist for dopamine D2 and D3 receptors vs. D4 and serotonin 5HT2 receptors (Mukherjee et al., 1999). Unlike benperidol, the D2-selective butyrophenone neuroleptic, this radiotracer also seems to be somewhat sensitive to endogenous dopamine levels (Mukherjee et al., 1997). The synthesis of [18F]fallypride is via a one-step nucleophilic substitution reaction of the corresponding propyl inflate with [18F]fluoride ion (Mukherjee et al., 1995). This reaction proceeded in 20% unconnected radiochemical yield to provide [18F]fallypride in high specific activity (900-1700 Ci/mmol) in a total synthesis time of only 60 minutes. Imaging data in primates for [18F]fallypride looks promising but no human data are yet available (Christian et al., 2000). Nicotinic receptor radiotracers A number of fluorine-18 labeled radiotracers for the nicotinic cholinergic receptor (nAChR) have beer, prepared, several of which are derivatives of the highly potent agonist, (-)-epibatidine (Liang et al., 1997; Ding et al., 1999; Marriere et al., 2000; Scheffel et al., 2000). All of these radiotracers have sub-nanomolar binding affinity for the nAChR which has lead to the difficulty that they are all extremely toxic, with LD50 values typically less than 1 umol/kg. This slim margin of safety has prevented use in human of even high specific activity radiotracers. This limitation has also applied thus far to carbon-11 labeled analogs. The most promising nAChR radiotracer developed to date is 2-[18F]fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine (2-18F-A-85380 or 2-[18F]FA). This compound is somewhat less toxic, having an LD50 in mice of "only" 15 umol/kg (Horti et al., 1998b), and fluorine-18 labeling via nucleophilic aromatic substitution on both the corresponding iodo (Horti et al., 1998a) and trimethylammonium triflate (Dolle et al., 1999) precursors has been reported. The latter synthesis provided 2-[18F]FA in 50% radiochemical yield (EOS) and a specific activity of >3000 Ci/mmol in a total synthesis time of 50 min. Whether the lower toxicity of 2-[18F]FA will allow evaluation of this radiotracer in humans remains to be seen.

EMERGING AREAS IN 18F-IMAGING Imaging gene therapy Several recent reviews have stressed the emerging importance of Nuclear Medicine imaging in pharmaceutical design. Pharmacokinetic experiments such as those described above for the fluoroquinolone antibiotics or competitive binding studies can provide direct in vivo information on the distribution and behavior of new pharmaceuticals. In the development of gene therapies, in vivo imaging may be the only adequate method for monitoring and evaluation of new techniques (Gambhir et al., 2000; Varagnolo et al., 2000; Wunderbaldinger et al., 2000). A more detailed discussion of imaging gene therapy can be found in this volume. The basic premise is to include the gene encoding a biochemical marker (such as an enzyme or receptor) in the same viral vector used to transfect the gene of interest for therapy. Thus imaging with a radiotracer specific for the marker provides an index of therapeutic gene incorporation. Markers currently

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under development for gene therapy monitoring are the herpes simplex virus type-1 thymidine kinase (HSVl-tk) and the dopamine D2 receptor. Several radiotracers for the HSVl-tk have been evaluated including 2'-deoxy-2'-[18F]fluoro-5-iodo-D-arabinofuranosyl-uracU ([18F]FIAU) and fluorine-18 analogs of the acycloguanosine derivatives 9-(3-[18F]fluoro-l-hydroxy-2-propoxy)methylguanine ([18F]FHPG), ganciclovir ([18F]FGCV) and penciclovir ([18F]FPCV) (for review see (Gambhir et al., 1999 and 2000) and references therein). As yet only one radiottacer for the D2 receptor, [18F]fluoroethylspiperone ([18F]FESP), has been evaluated for imaging gene incorporation. Labeled peptides As the role of bioactive peptides, such as endorphins, growth factors and other peptide hormones, has become better understood, interest has risen in developing peptide radiopharmaceuticals for in vivo imaging (Blok et al., 1999; Heppler et al., 2000; Varagnolo et al., 2000). Much of this interest has been fueled by oncology since many of the cell-surface receptors for peptide hormones are overexpressed in cancer. So far only a small number of fluorine-18 labeled peptides have been prepared. These include somatostatin analogs and [18F]fluoro-transferrin for imaging cell proliferation (Guhlke et al., 1994; Aloj et al., 1996; Wester et al., 1997), chemotactic peptides for imaging bacterial infection and possibly tumors (Vaidyanthan & Zalutsky, 1995), as well as fluorine-18 labeled antibody fragments (Page et al., 1994; Vaidyanthan & Zalutsky, 1994). Peptides can be radiolabeled in low specific activity by direct electrophilic [18F]fluorination on tyrosine residues (Labroo et al., 1991), however over the past decade a number of methods and reagents have been developed for labeling peptides with no-carrier-added fluorine-18. All of these latter methods involve conjugation of the peptide, usually by acylation, with a relatively small aryl[18F]fluoride. Labeling reagents include N-succinimidyl 4-[18F]fluorobenzoate (Vaidyanthan & Zalutsky, 1992; Vaidyanthan & Zalutsky, 1994), N-succinimidyl 4-([18F]fluoromethyl)-benzoate (Lang & Eckelman, 1994; Lang & Eckelma,n 1997), [18F]fluorobenzaldehydes (Herman et al., 1994), /V-succinimidyl 8-((4'-[18F]fluorobenzyl)amino)suberate (Page et al., 1994), and [18F]fluorophenacyl bromide (Kilbourn et al., 1987; Downer et al., 1997). A less successful method for photochemical conjugation using 4-azidophenacyl[18F]fluoride has also been reported (Wester et al., 1996). All of these methods are limited by possible effects of the labeled prosthetic group on the pharmacokinetics, receptor affinity and in vivo stability of the peptide in question. Hopefully, Nuclear Medicine will soon be able to take advantage of the continued technological advances in rapid, automated peptide synthesis methodologies used in biochemistry and molecular biology. In this way it may be possible to produce peptide radiopharmaceuticals directly from fluorine-18 labeled amino acids.

As discussed

previously, fluorine-18 labeled derivatives of all three aromatic amino acids are available as is 4[18F]fluoroproline (Hamacher & Stocklin, 1995; Wester et al., 1999a). A complete discussion of the use of labeled peptides as radiopharmaceuticals can be found elsewhere in this volume.

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REFERENCES Adam MJ and Jivan S (1988) Synthesis and purification of L-6-[18F]fluorodopa. Appl. Rod. Isot., 39, 12031210. Aloj L, Lang L, Jagoda E, Neumann RD and Eckelman WC (1996) Evaluation of human transferrin radiolabeled with N-succinimidyl 4([fluorine-18]fluoromethyl)benzoate. J. Nucl. Med., 37(8), 14081412. Arnett CD, Shiue CY, Wolf AP, Fowler JS, Logan J and Watanabe M (1985) Comparison of three 18Flabeled butyrophenone neuroleptic drugs in the baboon using positron emission tomography. J. Neurochem., 44(3), 835-844. Atkins HL, Christman DR, Fowler JS, Hauser W, Hoyte RM, Klopper JF, Lin SS and Wolf AP (1972) Organic radiopharmaceuticals labeled with isotopes of short half-life. V. l8F-Labeled 5- and 6fluorotryptophan. J. Nucl. Med., 13(10), 713-719. Babich JW, Rubin RH, Graham WA, Wilkinson RA, Vincent J and Fischman AJ (1996) 18F-Labeling and biodistribution of the novel fluoro-quinolone antimicrobial agent, Trovafloxacin (CP 99,219). Nucl. Med. & Biol., 23(8), 995–998. Banks WR, Borchert RD and Hwang D-R (1994) Improvements in the production of flourine-18 labeled butyrophenone neuroleptic agents. Appl. Rad. Isot., 45(1), 75–81. Barrio JR, Huang SC, Yu DC, Melega WP, Quintana J, Cherry SR, Jacobson A, Perlmutter MM, Namavari M, Satyamurthy N and Phelps ME (1996) Radiofluorinated L-m-tyrosines: new in vivo probes for central dopamine biochemistry. J. Cereb. Blood Flow and Metab., 16(4), 667-678. Bergman J and Solin O (1997) Fluorine-18-labeled fluorine gas for the synthesis of tracer molecules. Nucl. Med. & Biol., 24(7), 677-683. Block D, Coenen HH, Laufer P and Stocklin G (1986) N.C.A. (18F)-fluoroalkylation via nucleophilic fluorination of disubstituted alkanes and application to the preparation of N-[18F]fluoroemylspiperone. J. Labelled Comp..Radiopharm., 23, 1042. Blok D, Feitsma RIJ, Varmeij P and Pauwels EJK (1999) Peptide radiopharmaceuticals in nuclear medicine. Euro. J. Nucl. Med.,, 26(11), 1511–1519. Brodack JW, Kilbourn MR, Welch MJ and Katzenellenbogen JA (1986) NCA 16a-[18F]fluoroestradiol-17b: The effect of reaction vessel on fluorine-18 resolubilization, production yield, and effective specific activity. Appl. Rad. Isot., 37(3), 217–221. Brown WD, DeJesus OT, Pyzalski RW, Malischke L, Roberts AD, Shelton SE, Uno H, Houser WD, Nickles RJ and Holden JE (1999) Localization of trapping of 6-[18F]fluoro-L-m-tyrosine, an aromatic Lamino acid decarboxylase tracer for PET. Synapse, 34(2), 111–123. Buckman BO, Bonasera TA, Kirschbaum KS, Welch MJ and Katzenellenbogen JA (1995) Fluorine-18labeled progestin 16a,17a-dioxolanes: Development of high affinity ligands for the progesterone receptor with high in vivo target site selectivity. J. Med. Chem., 38, 328-337. Carroll FI, Scheffel U, Dannals RF, Boja JW and Kuhar MJ (1995) Development of imaging agents for the dopamine transporter. Medicinal Research Reviews, 15(5), 419–444. Chaly T, Bandyopadhyay D, Matacchieri R, Belakhleff A, Dhawan V, Takikawa S, Margouleff D and Eidelberg D (1994) A disposable synthetic unit for the preparation of 3-O-methyl-6-[18F]fluorodopa using a regioselective fluorodemercuration reaction. Appl.Rad. Isot., 45(1). 25–30.

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Lucignani G, Losa M, Moresco RM, DelSole A, Matarrese M, Bettinardi V, Mortini P, Giovanelli M and Fazio F (1997) Differentiation of clinically non-functioning pituitary adenomas from meningiomas and craniopharyngiomas by positron emission tomography with [18F]fluoroethylspiperone. Euro. J. Nucl. Med., 24(9), 1149–1155. Lui E, Chirakal R and Fimau G (1998) Enzymatic synthesis of (-)-6-[18F]fluoronorepinephrine from 6[l8F]fluorodopamine by dopamine b-hydroxylase. J. Labelled Comp. Radiopharm., 41, 503–521. Lundkvist C, Halldin C, Ginovart N, Swahn C-G and Farde L (1997) [18F]b-CIT-FP is superior to [ 1 1 C ] b CIT-FP for quantitation of the dopamine transporter. Nucl. Med. & Biol., 24(7), 621–627. Lundkvist C, Halldin C, Swahn CG, Hall H, Karlsson P, Nakashima Y, Wang S, Millius RA, Neumeyer JL and Farde L (1995) [O-methyl-11C]b-CIT-FP, a potential radioligand for quantitation of dopamine transporter. Preparation, autoradiography, metabolite studies, and positron emission tomography examinations. Nucl. Med. & Biol., 22, 905-913. Luxen A, Guillaume M, Melega WP, Pike VW, Solin O and Wagner R (1992) Production of 6-[18F]fluoroL-DOPA and its metabolism in vivo - a critical review. Nucl. Med. & Biol., 19(2), 149–158. Luxen A, Perlmutter M, Bida GT, van Moffaert G, Cook JS, Satyamurthy N, Phelps ME and Barrio JR (1990) Remote, semiautomated production of 6-[18F]fluoro-L-dopa for human studies with PET. Appl. Rad. Isot., 41(3), 275–281. MacFarlane JG, List SJ, Moldofsky H, Firnau G, Chen JJ, Szechtman H, Gamett S and Nahmias C (1997) Dopamine D2 receptors quantified in vivo in human narcolepsy. Biological Psychiatry, 41(3), 305310. Mach RH, Nader MA, Ehrenkaufer RL, Gage HD, Childers SR, Hodges LM, Hodges MM and Davies HML (2000) Fluorine-18-labeled tropane analogs for PET imaging studies of the dopamine transporter. Synapse, 37, 109–117. MacLaren DC, Gambhir SS, Satyamurthy N, Barrio JR, Sharfstein S, Toyokuni T, Wu L, Berk AJ, Cherry SR, Phelps ME and Herschman HR (1999) Repetitive, non-invasive imaging of the dopamine D2 receptor as a reporter gene in living animals. Gene Therapy, 6(5), 785–791. Mankoff DA, Tewson TJ and Eary JF (1997) Analysis of blood clearance and labeled metabolites for the estrogen receptor tracer [F-18]-16a-fluoroestradiol (FES). Nuclear Medicine & Biology, 24(4), 341– 348. Marriere E, Rouden J, Tadino V and Lasne M-C (2000) Synthesis of analogues of (-)-cytisine for in vivo studies of nicotinic receptors using positron emission tomography. Organic Letters, 2(8), 1121–1124. Maziere B, Coenen HH, Halldin C, Nagren K and Pike VW (1992) PET radioligands for dopamine receptors and re-uptake sites: chemistry and biochemistry. Nucl. Med. & Biol., 19(4), 497-512. Melega WP, Perlmutter MM, Luxen A, Nissenson CH, Grafton ST, Huang SC, Phelps ME and Barrio JR (1989) 4-[l8F]fluoro-L-m-tyrosine: an L-3,4-dihydroxyphenylalanine analog for probing presynaptic dopaminergic function with positron emission tomography. J. Neurochem., 53(1), 311–314. Mineura K, Sasajima T, Kowada M, Ogawa T, Hatazawa J and Uemura K (1997a) Early delineation of cerebral glioma using amino acid positron tracers. Computerized Medical Imaging and Graphics, 21(l),63-66.

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Mineura K, Shioya H, Kowada M and Uemura K (1997b) Tumor extent of slowly progressive oligodendroglioma determined by 18F-fluorophenylalanine positron emission tomography. Euro. J. Rad., 25(1), 30–35. Mislankar SG, Gildersleeve DL, Wieland DM, Massin CC, Mulholland GK and Toorongian SA (1988) 6(18F]Fluorornetaraminol. A radiotracer for in vivo mapping of adrenergic nerves of the heart. J. Med. Chem., 31(2), 362-366. Moerlein SM, Banks WR and Parkinson D (1992) Production of fluorine-18 labeled (3-yV-methyl)benperidol for PET investigation of cerebral dopaminergic receptor binding. Appl. Rad. Isot,, 43(7), 913-917. Moerlein SM and Perlmutter JS (1992) Specific binding of 3N-(2'-[18F]fluoroethyl)benperidol to primate cerebral dopaminergic D2 receptors demonstrated in vivo by PET. Neuroscience Letters, 148, 97-100. Moerlein SM, Perlmutter JS, Markham J and Welch MJ (1997) In vivo kinetics of [18F](Nmethyl)benperidol: a novel PET tracer for assessment of dopaminergic D2-like receptor binding. J. Cereb. Blood Flow and Metab., 17(8), 833-845. Mukherjee J, Yang Z-Y, Brown T, Lew R, Wernick M, Ouyang X, Yasillo N, Chen C-T, Mintzer R and Cooper M (1999) Preliminary assessment of extrastriatal dopamine D-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, 18F-fallypride. Nucl. Med. & Biol., 26, 519–527. Mukherjee J, Yang ZY, Das MK and Brown T (1995) Fluorinated benzamide neuroleptics-III. Development of (5)-N-[(l-allyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2,3dirnethoxybenzamide as an improved dopamine D-2 receptor tracer. Nucl. Med. & Biol., 22(3), 283296. Mukherjee J, Yang ZY, Lew R, Brown T, Kronmal S, Cooper MD and Seiden LS (1997) Evaluation of damphetamine effects on the binding of dopamine D-2 receptor radioligand, 18F-fallypride in nonhuman primates using positron emission tomography. Synapse, 27(1), 1–13. Murakami M, Takahashi K, Kondo Y, Mizusawa S, Nakamichi H, Sasaki H, Hagami E, Iida H, Kanno I, Miura S and Uemura K (1988a) The comparative synthesis of 18F-fluorophenylalanines by electrophilic substitution with 18F-F2 and 18F-AcOF. J. Labelled Comp. Radiopharm., 25(5), 573578. Murakami M, Takahashi K, Kondo Y, Mizusawa S, Nakamichi H, Sasaki H, Hagami E, Iida H, Kanno I, Miura S and Uemura K (1988b) 2-18F-phenylalanine and 3-18F-tyrosine—Synthesis and preliminary data of tracer kinetics. J. Labelled Comp. Radiopharm., 25(7), 773-782. Nahmias C, Wahl L, Chirakal R, Firnau G and Garnett ES (1995) A probe for intracerebral aromatic ammoacid decarboxylase activity: distribution and kinetics of [18F]6-fluoro-L-m-tyrosine in the human brain. Movement Disorders, 10(3), 298-304. Namavari M, Bishop A, Satyamurthy N, Bida G and Barrio JR (1992) Regioselective radiofluorodestannylation with [18F]F2 and [18F]CH3COOF: a high yield synthesis of 6[18F]fluoro-L-dopa. Appl. Rad. Isot., 43(8), 989-996. Namavari M, Satyamurthy N, Phelps ME and Barrio JR (1993) Synthesis of 6-[18F] and 4-[18F]fluoro-L-mtyrosines via regioselective radiofluorodestannylation. Appl. Rad. Isot., 44(3), 527-536. Namavari M, Styamurthy N and Barrio JR (1995) Synthesis of 6-[18F]fluorodopamine, 6-[18F]fluoro-mtyramine and 4-[18F]fluoro-m-tyramine. J. Labelled Comp. Radiopharm., 36(9), 825-833.

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Neumeyer JL, Wang S, Millius RA, Baldwin RM, Zea-Ponce Y, Hoffer PB, Sybirska E, Al-Tikriti M, Charney DS, Malison RT, Laruelle M and Innis RB (1991) [123I]-2b-Carbomethoxy-3b-(4iodophenyl)tropane: High-affinity SPECT radiotracer of monoamine reuptake sites in brain. J. Med. Chem., 34(10), 3144–3146. Oberdorfer F, Hofmann E and Maier-Borst W (1989) Preparation of 18F-labelled 5-fluorouracil of very high purity. J. Labelled Comp. Radiopharm., 27(2), 137–145. Ogawa T, Miura S, Murakami M, lida H, Hatazawa J, Inugami A, Kanno I, Yasui N, Sasajima T and Uemura K (1996) Quantitative evaluation of neutral amino acid transport in cerebral gliomas using positron emission tomography and fluorine-18 fluorophenylalanine. Euro. J. Nucl. Med., 23(8), 889-895. Paans AM, Pruim J, Smit GP, Visser G, Willemsen AT and Ullrich K (1996) Neurotransmitter positron emission tomographic-studies in adults with phenylketonuria, a pilot study. Euro. J. Fed., 155 (Suppl. 1), S78-S81. Page RL, Garg PK, Garg S, Archer GE, Bruland OS and Zalutsky MR (1994) PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody Fab fragment. J. Nucl. Med., 35(9), 1506– 1513. Palmer AJ and Widdowson DA (1979) The preparation of 18F-labelled 4-fluoroestrone and 4-fluoroestradiol. J. Labelled Comp. Radiopharm., 16, 14–16. Perlmutter JS, Stambuk MK, Markham J, Black KJ, McGee-Minnich L, Jankovic J and Moerlein SM (1998) Decreased [18F]spiperone binding in putamen in dystonia. Advances in Neurology, 78, 161–168. Perlmutter M, Satyamurthy N, Luxen A, Phelps ME and Barrio JR (1990) Synthesis of 4-[18F]fluoro-L-mtyrosine: a model analog for the in vivo assessment of central dopaminergic function. Appl. Rad. Isot., 41(9), 801-807. Petric A, Barrio JR, Namavari M, Huang S-C and Satyamurthy N (1999) Synthesis of 3b-(4[l8F]fluoromethylphenyl)- and 3b-(2-[18F]fluoromethylphenyl)tropane-2b-carboxylic acid methyl esters: New ligands for mapping brain dopamine transporter with positron emission tomography. Nucl. Med. & Biol., 26(5), 529-535. Pomper MG, Katzenellenbogen JA, Welch MJ, Brodack JW and Mathias CJ (1988) 21-[18F]Fluoro-16aethyl-19-norprogesterone: Synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography. J. Med. Chem., 31, 1360–1363. Pomper MG, VanBrocklin HF, Thieme AM, Thomas RD, Kiesewetter DO, Carlson KE, Mathias CJ, Welch MJ and Katzenellenbogen JA (1990) 11b-Methoxy, 11b-ethyl- and 17a-ethynyl-substituted 16aflurorestradiols: Receptor-based imaging agents with enhanced uptake efficiency and selectivity. J. Med. Chem., 33, 3143-3155. Reddy GN, Haeberli M, Beer H-F and Schubiger AP (1993) An improved synthesis of no-carrier-added (NCA) 6-[18F]fluoro-L-DOPA and its remote routine production for PET investigations of dopaminergic systems. Appl. Rad. Isot., 44(4), 645-649. Rinne JO, Bergman J, Ruottinen H, Haaparanta M, Eronen E, Oikonen V, Sonninen P and Solin O (1999) Striatal uptake of a novel PET ligand, [18F]b-CFT, is reduced in early Parkinson's disease. Synapse, 31,119–124. Romer J, Fuchtner F, Steinbach J and Johannsen B (1999) Automated production of 16a-[18F]fluoroestradiol for breast cancer imaging. Nucl. Med. & Biol., 26(4), 473-479.

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Romer J, Steinbach J and Kasch H (1996) Studies on the synthesis of 16a-[18F]fluoroestradiol. Appl. Rad. hot., 47(4), 395-399. Rosenspire KC, Haka MS, VanDort ME, Jewett DM, Gildersleeve DL, Schwaiger M and Wieland DM (1990) Synthesis and preliminary evaluation of carbon-11-raefa-hydroxyephedrine: A false transmitter agent for heart neuronal imaging. J. Nucl. Med., 31(8), 1328–1334. Ruth TJ and Adam MJ (1996) Synthesis of C-11 and F-18 labelled compounds for biomedical applications: Current status and challenges for the future. J. Rad.analyt. Nucl. Chem., 203(2), 457-469, Scheffel U, Horti AG, Koren AO, Ravert HT, Banta JP, Finley PA, London ED and Dannals RF (2000) 6[l8F]Fluoro-A-85380, an in vivo tracer for the nicotinic acetylcholine receptors. Nucl. Med. & Biol., 27(1), 51-56. Shiue C-Y, Bai Li, Teng R-R, Arnett CD and Wolf AP (1987) No-carrier-added N-(3[18F]fluoropropyl)spiroperidol: Biodistribution in mice and tomographic studies in a baboon. J. Nucl. Med., 28, 1164–1170. Stocklin G (1995) Fluorine-18 Compounds. In Principles of Nuclear Medicine. Wagner Jr HN, Szabo Z and Buchanan JW (eds), W.B. Saunders Co., Philadelphia, pp. 178–193. Stocklin GL (1998) Is there a future for clinical fluorine-18 radiopharmaceuticals (excluding FDG)? Euro. J. Nucl. Med., 25(12), 1612-1616. Stout D, Petric A, Satyamurthy N, Nguyen Q, Huang S-C, Namavari M and Barrio JR (1999) 2b1R carbomethoxy-3b-(4- and 2-[18 F]fluoromethylphenyl)tropanes: Specific probes for in vivo quantification of central dopamine transporter sites. Nucl. Med. & Biol, 26(8), 897-903. Suehiro M, Dannals RF, Scheffel U, Stathis M, Wilson AA, Ravert HT, Villemagne VL, Sanchez-Roa PM and Wagner, Jr. HN (1990) In vivo labeling of the dopamine D2 receptor with N[11C]methylbenperidol. J. Nucl. Med., 31(12), 2015–2021. Szajek LP, Channing MA and Eckelman WC (1998) Automated synthesis of 6-[18F]fluoro-L-DOPA using modified polystyrene supports with bound 6-mercuric DOPA precursors. Appl. Rad. Isot., 49(7), 795804. Tewson TJ, Yang D, Wong G, Macy D, DeJesus OJ, Nickles RJ, Perlman SB, Taylor M and Frank P (1996) The synthesis of fluorine-18 lomefloxacin and its preliminary use in human studies. Nucl. Med. & Biol., 23(6), 767-772. Tomiyoshi K, Amed K, Muhammad S, Higuchi T, Inoue T, Endo K and Yang D (1997) Synthesis of isomers of 18F-labelled amino acid radiopharmaceutical: Position 2- and 3-L-18F-a-methyltyrosine using separation and purification system. Nucl. Med. Comm., 18, 169-175. Vaalburg W, Coenen HH, Crouzel C, Elsinga PH, Langstrom B, Lemaire C and Meyer GJ (1992) Amino acids for the measurement of protein synthesis in vivo by PET. Nucl. Med. & Biol., 19(2), 227-237. Vaidyanthan G and Zalutsky MR (1992) Labeling proteins with fluorine-18 using N-succinimidyl 4[18F]fluorobenzoate. Nucl. Med. & Biol., 19(3), 275–281. Vaidyanthan G and Zalutsky MR (1994) Improved synthesis of N-succinimidyl 4-[18F]fluorobenzoate and its application to the labeling of a monoclonal antibody fragment. Bioconjugate Chemistry, 5(4), 352356. Vaidyanthan G and Zalutsky MR (1995) Fluorine-18 labeled chemotactic peptides: A potential approach for the PET imaging of bacterial infection. Nucl. Med. & Biol., 22(6), 759-764.

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VanBrocklin HF, Carlson KE, Welch MJ and Katzenellenbogen JA (1993a) 16b-([18F]Fluoro)estrogens: Systematic investigation of a new series of fluorine-18-labeled estrogens as potential imaging agents for estrogen-receptor-positive breast tumors. J. Med. Chem., 36, 1619–1629. VanBrocklin HF, Liu A, Welch MJ, ONeil JP and Katzenellenbogen JA (1994) The synthesis of 7a-methylsubstituted estrogens labeled with fluorine-18: Potential breast tumor imaging agents. Steroids, 59, 34-45. VanBrocklin HF, Pomper MG, Carlson KE, Welch MJ and Katzenellenbogen JA (1992) Preparation and evaluation of 17-ethynyl-substituted 16a-[18F]fluoroestradiols: Selective receptor-based PET imaging agents. Nucl. Med. & Biol., 19(3), 363–374. VanBrocklin HF, Rocque PA, Lee HV, Carlson KE, Katzenellenbogen JA and Welch MJ (1993b) 16b[l8F]Fluoromoxestrol: A potent, metabolically stable positron emission tomography imaging agent for estrogen receptor positive human breast tumors. Life Sciences, 53, 811–819. Varagnolo L, Stokkel MPM, Mazzi U and Pauwels EKJ (2000) 18F-Labeled radiopharmaceuticals for PET in oncology, excluding FDG. Nucl. Med. & Biol., 27(2), 103–112. Verhagen A, Studeny M, Luurtsema G, Visser GM, DeGoeij CCJ, Sluyser M, Niewberg OE, Van Der Ploeg E, Go KG and Vaalburg W (1994) Metabolism of a [18F]fluorine labeled progestin (21-[18F]fluoro16a-ethyl-19-norprogesterone) in humans: A clue for future investigations. Nucl. Med. & Biol., 21(7). 941-952. Vine EN, Young D, Vine WH and Wolf W (1979) An improved synthesis of 18F-5-fluorouracil. Intl J. Appl. Rad.Isot., 30, 401–405. Visser GWM, Gorree GCM, Braakhuis BJM and Herscheid JDM (1989) An optimized synthesis of 18Flabelled 5-fluorouracil and a reevaluation of its use as a prognostic agent. Euro. J. Nucl. Med.. 15, 225-229. Volkow ND, Wang GJ, Fowler JS, Logan J, Gatley SJ, MacGregor RR, Schlyer DJ, Hitzemann R and Wolf AP (1996) Measuring age-related changes in dopamine D2 receptors with 11 C-raclopride and 18F-Nmethylspiroperidol. Psychiatry Research, 67(1), 11–16. Wahl LM, Chen JJ, Thompson M, Chirakal R and Nahmias C (1999) The time course of metabolites in human plasma after 6-[18F]fluoro-L-m-tyrosine administration. Euro. J. Nucl. Med., 26(11), 1407– 1412. Weber WA, Wester HJ, Grosu AL, Herz M, Dzewas B, Feldman HJ, Molls M, Stocklin G and Schwaiger M (2000) O-(2-[18F]fluoroethyl)-L-tyrosine and L-[methyl-11C]methionine uptake in brain tumours: initial results of a comparative study. Euro. J. Nucl. Med., 27(5), 542–549. Wester H-J, Hamacher K and Stocklin G (1996) A comparative study of N.C.A. fluorine-18 labeling of proteins via acylation and photochemical conjugation. Nucl. Med. & Biol., 23(3), 365-372. Wester H-J, Herz M, Senekowitsch-Schmidtke R, Schwaiger M, Stocklin G and Hamacher K (1999a) Preclinical evaluation of 4-[18F]fluoroprolines: Diastereomeric effect on metabolism and uptake in mice. Nucl. Med. & Biol, 26(3), 259–265. Wester HJ, Brockmann J, Rosch F, Wutz W, Herzog H, Smith-Jones P, Stolz B, Bruns C and Stocklin G (1997) PET-pharmacokinetics of 18F-octreotide: A comparison with 67Ga-DFO- and 86Y-DTPAoctreotide. Nucl. Med. & Biol., 24(4), 275-286.

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7. PRODUCTION AND APPLICATION OF SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

RICHARD A. FERRIERI Brookhaven National Laboratory, Department of Chemistry, Upton, New York 119735000, NY, USA.

INTRODUCTION General Considerations in the Design of a PET Radiotracer Without a doubt, the success of PET as an imaging tool for basic research in the life sciences stems largely from the effort and success of chemists over the years in developing suitable radiotracers. This success derives in part from the fact that there exists today an extensive inventory of synthetic precursors, or small molecules labeled with short-lived positron emitting radionuclides, that can be used either for attaching radioactive isotopes to suitable substrates, or used as building blocks toward constructing larger biomolecules of interest. Since its infancy in the early 1960's, PET has evolved into a complex science for investigating the biochemical transformations of drugs and molecules within living systems. PET radiotracer chemistry too has evolved into a complex chemical science. Now radiotracers are engineered to be highly specialized probes for targeting specific regions such as neurotransmitter receptors, or chemical substances within the living system. In some instances this targeting can be as simple as measuring bioavailability. In others, it can become a more complex process of monitoring bioactivity of that region or substance. To keep pace with this growth, chemists are no longer driven solely by certain practical aspects of precursor production such as whether the precursor can be produced in amounts of radioactivity suitable for subsequent chemistry and final radiotracer formulation to meet the PET study protocol, whether it can be produced routinely over the course of the day, whether it is chemically and isotopically pure so as not to strongly influence subsequent chemistry and/or purification, or whether its specific activity is acceptable for the nature of the PET study. Now the design aspects of radiotracers for PET place additional demands on the chemist such as which radionuclide to choose to target a specific function (this is especially true in measurements of bioactivity), what position within the radiotracer to label, or which stereoisomer to use. (Langstrom et al, 1989a; Langstrom et al., 1989b; Langstrom et al., 1989c) Thus special emphasis has to be placed on the development of precursors that can satisfy all of these demands, and more as time goes on. A number of exhaustive reviews on the subject of precursor preparation have appeared in the literature over the years (Wolf et al., 1973; Clark & Buckingham 1975; Silvester, 1976; Wolf & Redvanly, 1977; Fowler &

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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Wolf, 1982; Ferrieri, 1983; Vaalburg & Paans, 1983; Langstrom et al., 1991; Fowler & Wolf, 1997). The interested reader, and especially newcomers, would certainly benefit from the insights of these earlier works. The scope of this chapter will encompass old, as well as the new approaches for conducting PET precursor preparation with the intent on being comprehensive without being exhaustive in the procedural descriptions. The hope, of course, is that this work provides sufficient insight as a general guide into methodologies with citations to appropriate references. It should also be noted that most of this chapter is dedicated to the subject area surrounding PET precursors labeled with carbon-11 and fluorine-18, since most of the milestones delineating biochemical transformations and movement of drugs within living systems have involved radiosyntheses using these radioisotopes. Short-Lived Positron Emitting Radionuclides for PET Without a doubt, the short-lived positron emitting radionuclides that have had the greatest impact in PET for radiotracer synthesis are carbon-11, nitrogen-13, oxygen-15 and fluorine-18. This is understandable in view of the fact that the first three of these isotopes are elements of life, and can be substituted for their stable counterparts without influencing the bioactivity of the molecule.

While fluorine-18 is not a significant

element in living systems, its half-life and properties makes its use in labeling of considerable value. Table 1 lists some of the physical properties of these radionuclides. Table 1. Physical Properties of the Short-Lived Positron Emitters Isotope

Half-life (min)

Specific Activity3

Maximum Energy (MeV)

Range (mm) in Water6

Decay Product

(Ci/mmol) Fluorine-18 Carbon-11 Nitrogen-13 Oxygen-15

110 20.4 9.96 2.1

1.71 x 106

0.635

6

0.96

7

1.19 1.72

9.22 x 10

1.89x 10 9.08 x 107

2.4 4.1 5.4 8.2

Oxygen-18 Boron -11 Carbon-13 Nitrogen-15

a.

Theoretical maximum specific activity; in practice, specific activities are typically 5000 times lower

b.

Maximum linear range.

because of unavoidable dilution with the stable element.

The development of any radiotracer for a PET study begins with the selection of an appropriate radionuclide. This becomes especially difficult when there exists multiple synthetic precursors that can allow chemists to produce the same, or nearly the same biomolecule, but with a different radioactive tag.

One example that

comes to mind is the radiolabeling of N-methylspiroperidol for measuring dopamine D2 receptors. This labeling has been performed with fluorine-18 in a multi-step two hour synthesis starting with [l8F]fluoride (Shiue et al., 1986), as well as with carbon-11 starting with [11C]H3I. (Wagner et al, 1983) Several factors can influence one's decision in this regard. The first is whether the physical half-life of the radioisotope matches the biological half-life of the process under investigation. Perhaps more important in the decision process is the precursor's specific activity. As Table 1 reveals, theoretical specific activities for these common radionuclides are varied. On the practical side, actual precursor specific activities are significantly lower owing to sources of endogenous stable isotopes in the materials used to construct the targets, the

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FLUORINE-18

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chemical materials irradiated in order to generate the radioisotope, the transfer lines that allow the radioisotope to be manipulated between the target and laboratory, as well as in the starting materials used for subsequent synthesis. Typically, carbon-11 specific activities tend to be higher than the other isotopes (Wolf & Redvanly, 1977; Finn et al, 1984; Fowler, 1986; Dannals et al., 1991) owing to the lower amounts of endogenous stable carbon in the starting materials.

Even so, it is important to realize that, since the

radiotracer is not free of carrier, the specific activity is changing proportional to the radioactive decay. Thus, the time necessary to prepare the synthetic precursor, and manipulate it through the subsequent synthetic pathways, and/or purifications can weigh heavily on one's decision. Finally, the nature of the information one is seeking from the PET measurement also plays an important role in the selection of the radioisotope. Whether one is seeking spatial distribution and regional concentrations of a target substance or neurotransmitter binding or uptake site, or whether one is seeking to assess bioactivity relying on metabolitic breakdown of the tracer could impact on this selection. General Methodologies for Producing Labeled Precursors Having addressed these important issues regarding the design of the radiotracer for the intended study, we now need to turn our attention to the actual stage of producing a useful synthetic precursor from what is typically a less useful source of the desired radionuclide. Historically, one can classify precursor preparation methods into those involving nonsynthetic approaches, and those involving more conventional synthetic approaches. The latter, of course, has received greater attention over the years perhaps owing to the fact that PET chemists are mostly derived from a synthetic organic chemistry background, and for whatever reasons, find greater security in developing more conventional synthetic approaches to doing things. In addition, such approaches are more readily automated as the chemical processing becomes standardized in the PET laboratory. This clearly becomes an issue when attempting to minimize radiation exposure to personnel. However, a brief discussion of nonsynthetic strategies to preparing precursors is warranted since, after all, PET's early roots in radiotracer development grew out of this area of research. Nonsynthetic approaches cover a rather broad area of radiochemistry that includes in-target or hot atom chemistry, radiation labeling, accelerated ion labeling, as well as labeling through the use of some excitation source of energy. Between 1950 and the mid-1970's, a number of chemists studied the chemistry of these short-lived positron emitting radionuclides as they were produced within the irradiation target as high energy atoms. This field became known as Hot Atom Chemistry, and flourished for a number of years under the aegis of basic energy science. Aside from the intrinsic value of understanding the basic chemical properties of these energetic or hot atoms, there was a strong commitment to providing a basic framework of knowledge that could allow chemists to control the chemical fate of these radioactive atoms within complex chemical environments. Such action set the early stage for producing the short-lived positron emitters in chemical forms that were useful for the synthesis of complex radiotracers. (Wolf & Redvanly, 1977; Ferrieri, 1983a) Unfortunately, chemists quickly realized that to produce sufficient quantities of radioactivity necessary for clinical research and application, the chemical fate of these primary hot atom products were often compromised by the harsh radiation field. A classic example of this behavior is the production of H[11C]N from a gaseous target comprised of 95% N2 and 5% H2. (Finn et al., 1971) The reader will later see that this is an extremely useful synthetic precursor for radiolabeling. At low irradiation doses, nucleogenic carbon-11 atoms produced from

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the 14N(p,a)11C reaction will react to form H[11C]N as the hot atom product. However, at the higher irradiation doses necessary to provide adequate levels of carbon-11 for a PET study, the intense ionizing radiation field caused by the higher flux of incident charged particles induces radiolytic reduction of this product to a less desirable form as [11C]H4. For the most part, the chemical form of the desired radionuclide, as it exits in the target, is usually the result of one or more physical and chemical changes occurring to the primary hot atom product, or more simply put the result of the radiation chemistry. It is interesting to note that even today, many of these phenomena are not entirely understood, but are relied on daily in the PET field for their ability to routinely produce sources of radionuclides. It is also interesting to note that chemists have had very little success at altering what goes on inside the production target, with the exception of a few cases, namely solid cryogenic targets, that will be discussed later under their appropriate subsections. Radiation labeling, as it is applied outside the target confines, is another area that has not been extensively exploited in the PET field for producing radiolabeled substances. Such labeling can be facilitated by either the introduction of an external electromagnetic radiation source, or by the internal radiation accompanying radioactive decay of the nuclide. The labeling effectiveness is attributed to the charge-state and/or excitation of the reactants rather than their kinetic energy. Effective use of radiation labeling lies in designing the chemical environment such that the resultant radiolytic species are selective in their reactions leading to a single labeled product. Unfortunately, application of this approach to preparing labeled compounds has been limited to carbon-14. An example of what could be accomplished with selective radiation labeling includes the preparation of 2,3-[14C]propanol by exposing [14C]ethylene and methanol to a gamma source. (Oae el al. 1968) Labeling by use of accelerated radioactive ions has also found limited application to preparing labeled compounds. (Wolf, 1960) Again, the majority of work utilizing this technique involved long-lived isotopes such as carbon-14 as 14C+, [14C]O+ and [14C]O2+ (Cacace et al. 1958; Pohlit et al., 1970; Lintermans et al. 1972), and tritium as T+ (Wolfgang et al., 1956). Finally, the use of external excitation sources such as electric discharge, microwave radiation or photosensitization have found application to producing labeled compounds through formation and reaction of radioactive ions or radicals. Unlike the other methods discussed, PET chemist have been successful at utilizing some of these strategies for producing PET precursors labeled with carbon-11, nitrogen-13 and fluorine-18. Examples include the production of ['1 'C]acetylene from [11C]H4 in an inductively coupled argon plasma (Crouzel et al., 1979), the production of [13N]H3 from [l3N]nitrogen gas using a microwave generated hydrogen plasma (Ferrieri, 1983b), and the production of [l8F]fluorine gas from electric anodic discharge. (Bergman et al., 1997) Precursors Labeled with Carbon-11 Nuclear Reactions for Producing Carbon-11 The nuclear reaction for producing carbon-11 that has had the greatest impact to PET has been the l4 N(p,a) H C reaction. This becomes clear for several reasons: (i) the radionculide can be produced from noncarbon material thus providing a source of high specific activity tracer, (ii) the reaction possesses a

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-IS

233

substantial nuclear cross-section of about 250 millibarns, thus affording substantial amounts of radioactivity from reasonable irradiations; and (iii) the reaction also possesses a relatively low threshold of 3.1 MeV, thus allowing production of radionuclide at reasonable particle energies. Other relevant reactions for producing carbon-11 are summarized in Table 2. Table 2, Nuclear Reactions for Producing Carbon-11 Q-Value

Threshold

Cross-

(MeV)

(MeV)

Section

Reaction

Particle

Reference

(mb)

7 P

I2

11

C(7,n) C

11

11

B(p,n) C

-18.7

18.7

-2.8

3.0

4 100

Hylten, 1970 Hintz & Ramsey, 1952

P P P

10

11

0

12

B(p,Y) C C(p,pn)11C

-18.7

20.3

l4

N(P,a)11C

2.9 6.5

d d

10

B(d,n)11C

d 3 He

12

11

11

B(d,2n) C

Crane & Lauritsen, 1934 Aamodt et al., 1952

3.1

100 250

-5.0

0 5.9

180 48

Brill &Sumin, 1960 Brill &

-20.9

24.4

Wilkinson, 1955

7.6

0

61 113

Epherre & Seide, 1971

Sumin, 1960

3

He 3 He 3 He 4 He 4 He

9

C(d,p2n)uC

Be(3He,pn)11C

10

B(3He,pn)11C

!!

3

-5.3

0 2.3 0 6.3

-13.0

18.8

-1.8

C(3He,4He)11C

1.9

B( He,p2n)

12

1.2

11C

16

4

4

11

0( He,2 He) C

9

Be(4He,2n)11C

285 35a 260 49 17

Hahn& Ricci, 1966 Brill, 1965 Brill, 1965 Brill, 1965 Brill & Sumin, 1960

4

10

4

11

He He 4 He

4

11

B( He,p2n) C

-19.6

27.4

B(4He,p3n)11C 12 C(4He,4He,n)11C

-31.1 -18.7

42.4 24.9

a

50 17a 48

Lindner & Osbourne, 1953

a.

Theoretical cross-section calculated according to statistical model. (Vaalburg and Paans, 1983)

Preparation of [11C]-Labeled Oxides The most widely used chemical form of carbon-11 for PET radiotracer synthesis is [11C]O2. Its widespread use is attributed to the fact that it can be easily harvested from the target gas stream either using liquid nitrogen cooled traps or even without the need for harsh cryogens using a mixture of reducing nickel catalyst

HANDBOOK OF RADIOPHARMACEUTICALS

234

and molecular sieve 5 A at ambient temeprature. In addition, virtually every synthetic carbon-11 precursor can be derived from this chemical form of carbon-11, as seen in the schematic below.

11 CH3SH (R)3P+11CH3I

CH3NO2

(NH2)2 CO

(CH3)2bo

R11CH2OH

n R CHO n R CH2Li

R

11

n

R CH2SH The production of [11C]O2 can be carried out utilizing continuous-flow or batch-wise irradiations of high pressure gaseous targets comprised of research grade N2 containing between 10 and 100 ppm levels of O2. As discussed earlier with regard to in-target chemistry, [11C]O2 is not the primary product, but the result of radiolytic oxidation of [11C]N radicals to [11C]O and eventually [11C]O2 under production conditions. (Christmanera/., 1975) [11C]O2 can also be produced from the proton or deuteron irradiation of solid enriched boron-10 targets, typically in the form of boron oxides. The advantage, of course, with the enriched boron targets is the zero energy threshold making them appealing for low energy high intensity proton accelerators. Typically, particle irradiation of a boron oxide target using an appropriate inert sweep gas can yield reasonable quantities of carbon-11, distributed between the products [11C]O, [11C]O2 and [11C]H4(Buckingham & Forse, 1963; Welch and Ter-Pergossian.,1968; Clark & Buckingham, 1971; Winstead et al., 1973; Ritchie, 1968: More & Troughton, 1972; Ferris et al. 1974) These targets tend to work best with a high power density of beam focussed onto the powdered matrix that causes a "quick melt" yielding a glassy structure. Complete conversion to [11C]O2 can be obtained by passing the target effluent gas through a copper oxide combustion furnace maintained at 800°C.

SYNTHETIC PRECURSORS LABELED WITH CARBON- 11 AND FLUORINE- 18

235

[11C]O is most often prepared through the catalytic reduction of [11C]02 over metallic zinc at 400°C. (Clark & Buckingham, 1975; Welch & Ter-Pergossian, 1968) The zinc catalyst is most effective when dispersed on an inert support. Asbestos has worked well for this application. This reduction is typically high, but not quantitative. However, unconverted [11C]O2 can be easily removed from the gas stream using Ascarite™ (silica supported LiOH) thus rendering the [11C]O in a pure form. Direct in-target production of usable quantities of [11C]O can be prepared as well using solid boron oxide targets. As described earlier, the same particle irradiations can be carried out with the exception that hydrogen gas is used instead of an inert gas to sweep the target matrix during bombardment. (Clark & Buckingham 1975; Winstead et al., 1973) This action results in 94% yields of the desired oxide which can be purified in much the same way as described above. Preparation of [11C] -Labeled Cyanides Carbon- 11 labeled cyanide as H[11C]N can be an extremely useful synthetic precursor for the PET chemist for replacing halogen atoms through nucleophilic substitution with the radiolabeled cyano group. It has been used in the synthesis of labeled amines, ketones, aldehydes, acids, and amino acids. (Fowler & Wolf, 1986) Over the years, several synthetic and nonsynthetic approaches have been explored for their ability to routinely prepare useful quantities of this precursor. Only a few are notable. (Finn et al. 1971; Lamb et al., 1971) Synthetic approaches for the preparation of H[11C]N rely on either [11C]O2 or [11C]H4as the starting material. One of the earliest methods involved the static reaction between [11C]O2 and potassium metal with carrier NH3. (Cramer & Kistiokosky, 1941; Loftfield, 1947; Lamb et al., 1971; Finn et al., 1971) The reaction tends to be messy requiring distillation of the precursor over sulfuric acid. 11

620°C

CO2 + 4K +- NH3 -

jj

*-

K CN + KH + 2KOH

The most widespread approach for preparing H[11C]N involves the catalytic conversion of [11C]H4 by reacting it with carrier NH3 over platinum metal at 1000°C. (Christman etal., 1975; Finn et al., 1971)

H11CN The conversion is typically 90% or greater for a single-pass flow reaction. The appeal of this approach is due to the fact that all the processing steps can be easily automated. Whether the chemist starts with11CH4 produced from the N2 + H2 gas target, or from [11C]O2 from the N2 + O2 target is a matter of preference. Some believe the [11C]H4 target provides a higher specific activity precursor that would extend to subsequent chemistry. In addition, macroscopic amounts of radiolytic NH3 are produced within the N2 + H2 target which can serve as the source of ammonia for the conversion to cyanide, thus eliminating the need to introduce an extraneous source of the gas. It should be noted, however, that trapping [11C]H4 on liquid nitrogen cooled molecular sieves can be problematic owing to the liquefaction of the target gas. Of course this is not an

236

HANDBOOK OF RADIOPHARMACEUTICALS

insurmountable problem, but it raises concern over certain safety issues. If one starts with [11C]O2 it can be readily trapped at ambient temperatures using a mixture of reducing nickel catalyst and molecular sieve 5A. Reduction to [11C]H4in a hydrogen atmosphere at 365°C is fast and quantitative. One precautionary measure should be noted. Macroscopic amounts of nitrogen oxides will form in the gaseous N2 + O2 targets when operated with high (>100 ppm) levels of O2. These oxides tend to trap on the nickel/molecular sieve, as well, giving rise to NH3 that can eventually poison the catalyst's reducing effectiveness. This has not been a problem for lower O2 levels. Several non-synthetic approaches for preparing H[11C]N have also been explored over the years. These included direct recoil labeling from proton irradiations of solid metallic cyanide targets, solid metallic amide targets (Lamb et al., 1971; Finn et al. 1971; Christman et al. 1970), as well as from gaseous targets comprised of mixtures of N2 and H2 (Christman et al., 1975; Lamb et al., 1971; Finn et al., 1971; Christman et al, 1970) 14

N(p,a)11C

n

NaCN

*»

14

N(p,a)11C

LiNH22

H2O

Na CN

11

+• H CN + NH3

While the NaCN target produces large amounts of carbon-11, there is an obvious constraint in the precursor's specific activity that hampers its use for PET. Recoil synthesis from LiNH2 is not any better owing to the low 3.6% yield of [11C]cyanide extracted. Gas targets comprised of 5% H2 and 95% N2 (O2 free) will also produce about a 50% yield of H[11C]N, with the remainder of the carbon-11 activity present as [11C]H4.

11 C*

+

N2

[11C-N=N]* [11CN] + H2 -

*»

*» [11CN] + N *• H11CN + H

Unfortunately, this method is not practical in that the product distribution is only reproducible at low irradiation doses where microcurie levels of the precursor are generated. Once higher irradiation doses are applied (>leV molecule-1 sec-1) to the target gas, near quantitative radiolytic reduction of the H[11C]N to [11C]H4 occurs thus requiring synthetic intervention in a post-irradiation treatment. More recently, cryogenic solid ammonia targets were investigated as a source for recoil labeling, and found to produce reasonable amounts of H[11C]N (30-40% of theoretical) even for high dose irradiations. (Firouzbakht et al.. 1999a) A

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18 comparison of quartz and silver target materials revealed that the silver target was less sensitive to applied dose relative to carbon-13 recovery as cyanide. Transition-mediated [11C]-cyanation of aryl rings is also noteworthy as a means to introduce carbon-11 into larger molecules. The usefulness of the technique was first realized using tricarbonylchromium complexes (Balatoni et al, 1989) and later with tetrakis(triphenylphosphine)palladium(0) 1994)

(Andersson & Langstrom,

These catalysts required special handling in order to exclude oxygen and water. More recently, it was

demonstrated that copper(I) salts will mediate a vast number of aromatic nucleophilic substitutions using [ II C]N. (Penchant et al, 1997). The copper salts have the advantage in that they don't suffer the instability problems of the chromium or palladium catalysts.

In addition, much of the chemistry generating the

[11C]aryl nitriles, and their subsequent conversion to other functional groups can be carried out as single-pot reactions.

11

H CN

11 Cu CN

Cuso4 go

Na2S2O5

>

n

Cu CN

+

(R= H, 2 -NO2 , 2 -NH2 , 3 - OCH3 , 4 - Br)

H

Within the context of this section it is worth noting how one can introduce other functional groups into the labeled cyanide precursor thus producing a new line of precursors with multifunctional properties that can serve to increase the diversity of molecular structures possible for rapid labeling synthesis. For example,

238

HANDBOOK OF RADIOPHARMACEUTICALS

substitution between [11C]N~ and corresponding dibromo-, diiodo- and ditosyl- compounds using Kryptofix 2.2.2 as an anion activator will produce 80-95% yields of the corresponding radiolabeled halonitriles.

X-(CH2)n-Y

+ CN' -

» X-(CH2)n CN

X, Y= I, Br, tosyl

In one example cited, 4-iodobutyro[CN-11C]nitrile was used to alkylate an achiral glycine derivative producing DL-[6-11C]lysine (Antoni et al., 1989). In yet another example, [CN-11C]acrylonitrile can be prepared in 35% yields from the substitution reaction between [11C]N and vinylbromide catalysed by tetrakis(triphenylphosphine)palladium (Antoni et al. 1991). TT n

H

n

11 CN

H

to crown A Pd[P(Ph)3]4

Br

H

H

/

\n

H

CN

This precursor affords the synthetic opportunity to introduce a radiolabeled cyanoethyl group into a larger molecule. It is also worth mentioning the development of an unusual precursor, [!1C]cyanogen bromide. Unlike hydrogen cyanide or any of the other nitriles previously mentioned, cyanogen bromide possesses a reversal of polarity, thus offering the cyano group as an electrophilic reagent. [11C]Cyanogen bromide is readily prepared in 95% radiochemical yields from H[11C]N using a simple solid-phase on-line procedure that involves passing the H[11C]N through a tube containing pyridinium tribromide and antimony powder (Westerberg & Langstrom, 1997).

11 H CN

Pyridium Tribromide

Sb

*•

11 CNBr

This precursor can allow chemists an opportunity to achieve a variety of useful functional group transformations yielding radiolabelled cyanates, thiocyanates and cyanamides (Westerberg & Langstrom, 1993).

11 /-IXTTJ,CIS Hi

R3NH ,.„

W »•

11 . n?^M p\n~nRr ~

RSH

4 n » I? ! (V K.O ^

RNH

2

^XTTT

11 CN

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-IS

239

Preparation of [11C]-Labeled Methylating Agents Although some of the earliest syntheses with carbon-11 depended directly on radiolabeled carbon dioxide and cyanide (Fowler & Wolf. 1986) chemists today tend to rely on [11C]H3I as the precursor of choice for introducing carbon-11 to organic molecules. (Langstrom and Lundqvist, 1976; Fowler & Wolf, 1982) This is with good reason as there exists today several commercial systems that will automatically process [11C]O2 and generate batches of [11C]H3I for the chemist. Carbon-11 labeled methyl iodide can be prepared by both synthetic and nonsynthetic approaches. The most common preparative method depends on the reduction of [11C]O2 to [11C]H3OH by LiAlH4 followed by subsequent iodination using hydroiodic acid. (Langstrom & Lundqvist, 1976; Marazano et al., 1977; Iwata et al., 1979) 11 CO2

LiAlH4 u »* CH3OH

HI -

il >• CH3I

These steps can be easily carried out in a single-pot reactor. However, some precautionary measures should be noted. After the initial trapping of the [11C]O2 in the LiAlH4, the solvent, which is typically tetrahydrofuran, requires removal using vacuum and heat. Very little loss of carbon-11 is seen during this step, as the activity remains complexed within the lithium salt. However, the salt must be cooled again prior to adding the concentrated hydroiodic acid, or else the exothermicity of the reaction could result in an explosion. Once added, the mixture is again heated to reflux thus allowing the [11C]H3I to be distilled off in an inert gas stream. Typically, conversion of [11C]O2 to 11CH3I is fast and efficient resulting in greater than 80% yields of the precursor within 5 to 10 minutes. Of course, the biggest issue is how to maintain some sense of control over the precursor's specific activity. This is especially critical when manipulating LiAlH4 as it will readily absorb carrier CO2 from exposure to air. Special attention must be given to maintaining an inert environment at all times. Typically, precursor specific activities in the range of 1 to 3 Ci/u;mole are attainable. Another method that has gained recent popularity relies on a gas-phase synthesis involving [11C]H4 and I2 (Larsen et al. 1997; Link et al., 1997). The process can begin with either [11C]H4 that is produced directly within the target, or with [11C]O2 which must then be reduced to [11C]H4 over nickel at 365°C.

720 c

I Ti 1*

'1

11 .r'TT.

11

° > . 2T-

f^TT L.J.14

+

T..

11 . .PHo + HT

te 9

— te.

11

r'T-r.T a.

The [11C]H4 is passed through a quartz tube at 720°C which contains I2 vapor. The high temperature dissociates the iodine molecule to generate iodine atoms that are free to abstract hydrogen from an [11C]H4 molecule. The resulting [11C]H3 radical, in turn, attacks another I2 molecule yielding [11C]H3I The

240

HANDBOOK OF RADIOPHARMACEUTICALS

disadvantage of this approach is that the thermolysis induced radical reaction is not terribly efficient, and so, the gas containing the reactants must be recirculated several times through the furnace. Nonetheless, the synthesized [11C]H3I can be easily harvested from this recirculated gas stream using porous polymer supports like Porapak N or Porapak Q, and later released for subsequent synthesis. Typically, 40% of the carbon-11 is converted to [11C]H3I with 15 minutes of processing. The yield is more than adequate to produce several hundred millicuries of [11C]H3I on a per batch basis, with a specific activity of between 6 and 8 Ci/p.mole, corrected to end-of-bombardment. Key advantages of this approach are that it is easily automated, and the precursor's specific activity typically exceeds that obtained by the "wet" chemistry method. GE Medical Systems, Inc. (Husbyborg 752 29 Uppsala, Sweden) presently markets a fully automated system of this approach. [11C]Methyl iodide can also be prepared by recoil labeling through the proton irradiation of gaseous targets comprised of N2 and 10% HI (Wagner et al., 1981). Unfortunately, the precursor yield is only 27% at low irradiation doses, and is dose sensitive. Attempts to minimize radiolytic destruction of the [11C]H3I using a high flow of target gas were unsuccessful in making this target practical for producing large amounts of radioactivity. Interestingly enough, this approach does possess potential for producing the highest specific activity of all the methods described. However, the inherent problems associated with manipulating the corrosive target gas, as well as dealing with a low yield of radiolabeled precursor that requires rigorous online purification, far outweigh this advantage. Two other radiolabeled methylating agents are worth mentioning within the scope of this section as they represent attempts to create a more reactive precursor thus allowing chemists to perform methylations under milder conditions. Carbon-11 labeled methyl lithium is one example of this. This precursor can be prepared by an equilibrium reaction between n-butyl lithium and [11C]H3I (Reiffers et al., 1979; Reiffers et al., 1980).

n-BuLi

+

11

CH3I

11

>• CH3Li + BuI

Typically, the interconversion is carried out at low temperature with excess n-butyl lithium to drive the reaction towards the [11C]H3I side, while at the same time avoiding unwanted coupling. Interconversions are nearly complete within 10 minutes yielding specific activities for the [11C]H3Li that are comparable to that of the starting material, [11C]H3I. The downside of this process is that a large amount of n-butyl lithium is present which can influence subsequent synthetic steps. Carbon-11 methyl trifluoromethanesulfonate (methyl triflate) is another example of preparing a more reactive agent that allows chemists an opportunity to carry out alkylations under milder conditions (Jewett, 1992).

11

CH3I

AgOSO2CF3 11 —^ -+ CH3OSO2CF3 150 C

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

241

The process for preparing this useful precursor begins by passing [11C]H3I through a small soda-glass column containing silver triflate -impregnated graphitized carbon. The conversion to [11C]methyl Inflate is fast and efficient at 150-200°C, with the precursor readily trapped at 0°C at the outlet stream. Finally, the [11C]-labeled Grignard reagent methyl magnesium iodide should be mentioned for its versatility in 1,2-carbonyl additions yielding [11C]-N-tert-butyl group on a larger molecule, and in the synthesis of [11C]-sec-alcohols. (Elsinga et al., 1995) nu T

CH3I

™

diethylether, I2

+ Mg

—

u

+•

O

CH3MgI

OH 11

CH3MgI

H R = Ph, PhCH2CH2 The process involves reaction of [11C]H3I with magnesium turnings mixed with iodobenzene in ether. The organic halide is an essential component toward initiating the Grignard reaction. Typically, conversions in the range of 60% to [11C]methyl magnesium iodide are obtainable.

Preparation of [11C]Formaldehyde Like many of the other carbon-11 precursors, [11C]formaldehyde ([11C]H2O) has its utility for allowing chemists to carry out [11C]-carbonylation reactions. Many procedures have been established to produce [11C]H2O. The methods previously described typically require a two-step process involving reduction of target-produced [11C]O2 to [11C]H3OH using lithium aluminum hydride, followed by oxidation of the [11C]HsOH to [!1C]H2O on metallic converters and catalysts such as silver wool (Marazano et al., 1977) or ferric molybdenum oxide (Christman et al., 1972; Straatman & Welch, 1975). U

CO2

LiAlH4

u

*» CH33OH

Fe-Mo Catalyst

o-

580C

11

*> H CHO

Two other solution-phase approaches are worth mentioning. The aqueous-phase oxidation of [11C]alcohols to [11C]aldehydes using tetrabutylammonium bichromate can be successful, but is limited to subsequent chemistry tolerant of the aqueous environment (Halldin & Langstrom, 1984). Another recent approach relies on the direct reduction of [11C]O2 to [11C]H2O using metal hydrides at low temperature (Nader et al., 1997). Typically, 58% yields of [11C]H2O can be attained using lithium aluminum hydride in tetrahydrofuran at 50°C. The nature of the metal hydride, as well as the solvent temperature are key to optimizing the

HANDBOOK OF RADIOPHARMACEUTICALS

242

reduction. For instance the radiochemical purity of the precursor drops significantly above -45°C due to large yields of [11C]H3OH produced in the process. Preparation of [11C]Phosgene As an acid chloride, [11C]phosgene ([11C]OC12) opens up many possibilities for preparation of other useful precursors. The preparation of anhydrous [11C]urea from [11C]OC12 is one such example where the [11C]urea precursor can be subsequently used to synthesize 2-[11C]thymidine (Steel et al., 1993; Steel et al., 1999). Other possibilities include the synthesis of [11C]alkyl carbonates from reaction of 11COC12 with alcohol, or the synthesis of [11C]alkyl carbamates from combined reaction with alcohol and ammonia. 11

NH Liq. coci2 —^-V o=n_/ c(

NH2

3

N

NH?

H2SO4, SO3 EtOH

NH2 OH

2 - ' Deoxyribose -1 - phosphate

HO

Thymidine phosphorylase

OH 2-[ CJThymine

2-[11C]Thymidine

Perhaps one of the oldest methods for preparing [11C]OC12 involves the ultraviolet photochemical reaction between [11C]O and C12 (Brinkman et al., 1978; Christman et al., 1979; Roeda and Western, 1981). The conversion is fast and quantitative when conducted under static conditions using excess C12 gas. 11

CH,

C12

fry

COC12

In addition, the [11C]OC12 is readily purified by passing the ampoule contents over antimony metal to remove the excess Cl2.

SYNTHETIC PRECURSORS LABELED WITH CARBON- 11 AND

FLUORINE-18

243

Another method that relies on [11C]O involves a catalytic chlorination reaction. (Roeda et al. 1978) Typically, [11C]O is produced through reduction of [11C]O2 over hot zinc, as described earlier in this chapter. The gas effluent from this step is simply passed through a second furnace at 280°C containing PtCl4. 11

zinc asbestos

-

-

\\

-

PtCl4 C

°

280V

The conversion .of [11C]O-to-[11C]OCl2 is usually 60%, and while less than the photochemical reaction, does offer the advantage of a flow system for manipulating the radioactive substances. One other approach is worth mentioning because of its ability to produce large amounts of [11C]OC12 relatively quickly. This method involves a two-step process. First, [11C]H4 is converted to [11C]Cl4 through reaction with Cl2 gas at 390°C. The conversion under flow conditions is typically fast and in under 10 minutes, providing a 70% yield of [11C]C14. The source of [11C]H4 can either be from the N2 + H2 gas target where C12 gas is mixed with the target gas during unloading. Another way is to start with [11C]O2 produced from the N2 + O2 target. The advantage here is that the [11C]O2 can be easily trapped during target unloading on molecular sieve/reduced nickel at ambient temperature, reduced to [11C]H4 and then slowly flowed through the chlorination furnace. Either source of carbon-H will suffice. The [11C]C14 produced in the chlorination step is then mixed in a stream of O2 gas, and passed through a second furnace at 300°C containing iron granules (Steel et al., 1999). 390

°C

11

Fe/02

- cci4 -*-

,,

coci2

The conversion of 11CCl4 to 11COC12 is about 79% and takes only about 3 minutes to complete. The 11COC12 can be easily purified online by passing the gas effluent through metallic antimony. Preparation of C2 and Larger [11C]Alkyl Iodides Carbon-11 labeled alkyl iodides such as ethyl, propyl, and butyl iodides are useful precursors that enable the chemist the ability to extend the size of the side alkyl chain beyond that of a simple methyl group. (Langstrom et al., 1986) This is sometimes desirable when designing the radiotracer with the radioactive label on different positions of the molecule. One application of this is the radiolabeling of N,N-dipropyl-2|4~methoxy-3-(2-phenylethoxy)phenyl]ethylamine (NE-100) in two different positions by alkylating the Ndespropyl precursor with [11C]propyl iodide, and the O-desmethyl precursor with [11C] methyl iodide (Ishiwata et al., 1998). Another reason for adding these alky] iodides to the arsenal of labeling precursors is that they provide a useful springboard for generating precursors with other functionalities. The standard approach for preparing [11C]alkyl iodides consists of the reaction of a Grignard reagent with [11C]O.2 followed by reduction using lithium aluminum hydride and finally reaction with aqueous hydroiodic acid. A drawback to this approach is that it will produce [11C]H3I as a by-product. Unfortunately, [11C]H3I is more reactive than any of the higher-order akyl iodides, and thus poses problems by potentially hindering the radiolabeling effectiveness of the desired alkyl iodide precursor, as well as in producing unwanted

244

HANDBOOK OF RADIOPHARMACEUTICALS

radiolabeled by-products that necessitate more complex purification schemes. The former issue is especially noteworthy in view of the fact that larger amounts of carrier CH3I mass are produced in the process owing to CO2 contamination of the lithium aluminum hydride. Chemists typically address these issues by designing an additional purification step into the synthetic scheme prior to reaction with the labeling substrate. One approach that has been extremely successful, owing to the volatility of the alkyl iodide precursors, is the use of gas chromatography. While this may sound complicated in the normal scheme of things, it can be a rather simply solution to the problem and amenable to automation. (Ishiwata et al., 1999) Radiochemical yields after gas chromatography were on the order of 27%, 22% and 12% for [11C]ethyl iodide, [1!C]propyl iodide and [11C]butyl iodide, respectively, with preparation times increasing proportionally with the size of the alkylating agent from 12 to 19 minutes. Preparation of [11C]Nitroalkanes The development of [11C]-labeled nitroalkanes as a class of synthetic precursors occurred out of necessity to increase flexibility in radiochemistry (Schoeps et al., 1989; Schoeps et al., 1991). Nitroalkanes are a versatile class of labeling precursor in the sense that they can be readily converted into carbon nucleophiles by the addition of base. Their reactions through nucleophilic substitution and aldehyde condensation are well documented (Mathieu and Weill-Raynal, 1973). Once reacted, the nitro group can be easily converted to other functionalities such as a carbonyl group (Nef's reaction) or reduced to an amine.

i)OH2) H2SO4

CHO I R

OH

CHO I JtltAJrl 1 R

CH3NO2 U

1

R

LiAlH4

^

CH2NH2 urr-rnj

R

The synthetic importance of this class of labeling precursor has been demonstrated using [11C]nitromethane in the synthesis of [11C]phenethylamine (Schoeps & Halldin, 1992) and [11C]dopamine (Schoeps et al., 1993) via condensation with the appropriate aldehydes to yield the corresponding [11C]nitrostyrenes.

CH3NO2

CHO

CH=CH-N02

!)NaOH

2)HC1

LiAlH4

CH=CH-NO,

NH-

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

245

More recently, the utility of this class of precursor was extended by developing a strategy where the [11C] Pnitrophenethyl alcohols could be obtained in preference to the styrene product through condensation of [11C]nitromethane with various substituted benzaldehydes using tetrabutylammonium fluoride (TBAF) as a catalyst. This strategy can then allow for the preparation of [11C]-labeled phenylethanolamines such as norphenylephrine and norepinephrine (Nagren et al., 1994).

CHO

CH3NO2

* " \J 1 / ' OH HO

HO

Raney Ni Formic Acid

ii

CH— CH2-NH2

OH Nitroalkanes labeled with carbon-11 can be easily prepared by reacting the appropriate [11C]alkyl iodide with silver nitrite at 80°C. AgN02

R-CH2I

R-11CH2NO2

80°C R = H, CH3,

This approach is amenable to on-line processing where the purified [11C]alkyl iodide is flowed in a nitrogen or helium stream through a 3 mm id x 4 cm length soda glass column packed with 0.4 g of silver nitrite at 2030 mL/min. (Schoeps et al., 1989) Radiochemical yields (based on 11CO2) of 55%, 30% and 40% are typical for preparing [11C]nitromethane, [11C]nitroethane and [11C]nitropropane, respectively.

Preparation of [11C]Alkylthiols In yet another class of precursor that includes the [11C]alkylthiols, the chemist has the ability to carry out Salkylation reactions. One area where these precursors have been useful is in the enzymatic synthesis of 2amino-4-([11C]methylthio)butyric acid ([11C]methionine) and its derivatives using immobilized y-cyano-aaminobutyric acid synthase. [11C]Methionine is widely used for clinical PET studies on amino acid metabolism in tumors (Strauss & Conti, 1991; Koh et al., 1994; Leskinen et al., 1997) as well as in the brain. (Bustany, 1983; O'Tauma et al., 1991; Salmon et al, 1996)

246

HANDBOOK OF RADIOPHARMACEUTICALS

Synthetically, the [11C]alkylthiols derive from a pure source of [11C]alkyl iodides (Suehiro el at., 1995; Kaneko et al., 1999). A number of synthetic methodolgogies have been tested including the use of heated reaction tubes. By far, the best procedure involves trapping the purified alkyl iodides in a 0.2 mL solution of DMF containing 2 mg of NaSH. The contents are then heated to 120°C, and the [11C]alkylthiols are immediately transferred under a nitrogen or helium gas stream to a second vessel where they can react. Typically, this approach will produce radiochemical yields of 91%, 92% and 98% for [11C]methanethiol, [11C]ethanethiol and [11C]propanethiol, respectively. Interestingly enough, reaction efficiencies are extremely sensitive to the level of O2 dissolved in the DMF solvent. Yields decrease when too little or too much O2 is present. The exact reason for this behavior is not clear. However, peak performance for preparing these precursors appears to occur when the NaSH is dissolved in the DMF at room temperature approximately 30 to 40 minutes prior to use. Preparation of [11C]Urea Over the years, [11C]urea has received considerable attention owing to its use in synthesizing 2-[11C]thymidine, a radiotracer that has been investigated for in vivo monitoring of cell proliferation in tumors using PET (Van der Borght et al., 1991; Labar & Van der Borght, 1991). The development of a tracer to monitor DNA synthesis has far reaching applications for the investigation of both tumor growth and response to anti-proliferation therapies, although the use of 2-[11C]-thymidine is limited by the presence of labeled metabolites (Van der Borght et al., 1990; Shields et al., 1990; Shields, 1993). Two methodologies for preparing usable quantities of [11C]urea are available. These approaches are depicted below. 11

CH

4

+

Pt

NH3

n

KMnO4

n

(NH3)2SO4

mono » H CN ——-+• KO CN •

o=c; 4

+

C12

390 °C

^

11

CC14

Fe/02

-^r-*>

NH

„

2

COC12

The first approach utilizes H[11C]N (Emran et al., 1985; Link et al., 1995). Several schemes have already been discussed for the preparation of H[11C]N. The formation of urea from this precursor begins by converting it to [11C]NH4CN. This is accomplished by collecting the H[11C]N in 0.2 mL of KMnO4 (0.032 M) containing 0.05 mL KOH (2M). The 11 CN/MnO 4 ~ mixture is heated to 100°C after which 0.2 mL (NH4)SO4 (0.75 M) and 0.1 mL ethanol are added. The mixture is again heated to 170°C for 3 minutes. Separation of insoluble MnO2 from the reaction mixture is accomplished by filtering through a disposable column possessing a 0.45 u.m filter. The alcoholic urea mixture must then be dried prior to further reaction. Radiochemical yields are typically greater than 35%. A second approach to preparing anhydrous [11C]urea utilizes [11C]phosgene (Steel et al., 1993). Like H[11C]N, there are several methods to preparing the starting precursor, [11C]phosgene, all of which have been

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND

FLUORINE-18

247

discussed earlier. The [11C]phosgene, as it is produced on-line, is mixed with a stream of oxygen at 10 niL/min and passed through a vessel containing 300 |jL of liquid ammonia maintained at -33°C. After about 5 minutes, the vessel is warmed to remove the ammonia thus allowing the [11C]urea to be taken up in a suitable anhydrous solvent. Total radiochemical yield by this approach is about 30%. This approach does offer a slight advantage in that it appears easier to automate (Steel et al, 1999). Preparation of [11C]Acetone Carbon-11 labeled acetone is a useful precursor in the synthesis of radiolabeled compounds containing isopropyl (Berridge et al., 1992; Rubottom & Kim, 1983) or acetonide functions (Berridge et al., 1994). Most recently, [11C]procaterol, a p2-adrenoceptor agonist, has been radiolabeled for PET using this precuror. (Visser et al., 2000) The general approach to preparing [11C]acetone is through reaction of [11C]O2 with methyl lithium (Berger et al., 1980). Typically, the organolithium reagent is present in large excess relative to the no-carrier-added concentrations of [11C]O2. This aspect has a downside in that large amounts of [11C]tert-butanol are also produced at the expense of [11C]acetone. MeLi

O

MeLi

LiO

QJJ

quenching agent

However, a slight modification to this methodology includes introducing diphenylamine to selectively quench the excess organolithium reagent prior to hydrolysis of the acetone diolate intermediate (Studenov et al., 1999). This modification allows for 100% radiochemical yields of [11C]acetone to be produced. Preparation of [11C]-Labeled Phosphonium Salts used as Wittig Reagents The Wittig reaction is typically used for extending carbon chains with one or more carbon atoms by converting aldehydes and ketones into alkenes (Maryanoff & Reitz, 1989). This reaction has been applied to the carbon-11 radiolabelling of several terminal and branch-chained alkenes (Kihlberg et al., 1990; Grierson et al,, 1993), and more recently improved upon by utilizing polymer-bound reagents (Ogren et al., 1995).

248

HANDBOOK OF RADIOPHARMACEUTICALS —CH—CH 2 CH=CH2

In typical reactions, [11C]H3I is trapped at ambient temperature in a solvent solution of 10% tetrahydrofuran and 0-dichlorobenzene containing 3 imol of the polymer-bound triphenylphosphine.

Polystyrene cross-

linked with 2% divinylbenzene works well as the polymer support After trapping, the mixture is heated to 160°C for 3 minutes to allow the radiolabeled phosphonium salt to form. Once cooled, 0.20 mmol of the appropriate aldehyde in o-dichlorobenzene solvent is added and the reaction is again heated for 3 minutes at 160°C. Radiochemical yields ranging from 29-65% are typical depending on the nature of the aldehyde substrate. Precursors Labeled with Fluorine-18 Nuclear Reactions for Producing Fluorine-18 Unlike carbon-11, fluorine-18 possesses a much lower positron energy (a maximum range of 2.4 mm) thus making it a very attractive radioisotope for localization measurements requiring high-resolution PET. An additional advantage is that it possesses a significantly longer half-life (110 min) than carbon-11 thus affording the radiochemist additional time to perform more complex synthetic manipulations. Over the years a number of nuclear reactions have been explored for their efficacy in generating synthetically useful quantities of fluorine-18. Table 3 lists these reactions along with pertinent nuclear data.

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

249

Table 3. Nuclear Reactions For The Production of Fluorine-18 Particle

Reaction

Q-Value (MeV)

Threshold Cross (MeV) Section

Reference

.^y.y™^~,~^,,^™-^~,,r~Y"".~..-,. -•—-•—-

t 3

He

3

I5

0(t,n)11JF 16 O(3He,p)18F

O(3He,n)18Ne-V8F

1.270 2.003

0

-3.196

3.795

400

He

I6

a

16

O(a,d)18F

-18.544

23.180

260

a

16

0(a,2n)18Ne-»18F

-23.773

29.716

—

20

2.796

0

3

20

-2.697

3.102

3

20

-7.926

9.115

—

-2.436

2.571

700

Ne(d, a)18F

He

He

Ne(3He, ap) 18F Ne(3He,an)18Ne-y18F

115

VeraRuiz, 1988 Nozaki et al., 1974; Fitschen et al, 1977 Nozaki et al., 1974; Knust and Machulla, 1983 Clark and Silvester, 1966 Nozaki et al,, 1974 Helus et al., 1979; Casella et al., 1980; Blessing et al., 1986 Backhausen et al., 1981 Backhausen, et al., 1981;Crouzel and Comar, 1978 Ruth and Wolf,1979; /., 1984

In the early days, fluorine-18 was a reactor produced radioisotope requiring a rather complicated two-step process involving fast neutron bombardment of a solid lithium-6 target to generate the requisite tritons to drive the l6O(t,n)18F reaction. Issues regarding tritium contamination within the beam-line as well as within the target never made this a practical approach. Today, cyclotron production is clearly the method of choice owing to the greater simplicity of the target designs, as well as the over-all higher yields of the radioisotope. The most commonly used nuclear reactions to produce fluorine-18 include the 18O(p,n)18F and 20Ne(d,a)18F reactions with the proton bombardment on enriched oxygen-18 providing significantly higher yields and improved precursor specific activity. (Ruth & Wolf, 1979) One's decision in selecting a particular method over another for production is contingent on several factors including: (i) whether the available cyclotron is capable of multiple particles; (ii) whether it is desirable for the fluorine-18 source to be aqueous or

250

HANDBOOK OF RADIOPHARMACEUTICALS

anhydrous; (iii) whether the radiolabeled precursor needs to be nucleophilic or electrophilic; and (iv) whether that precursor needs to possess a high specific activity. As a general rule to follow, proton irradiation of enriched water will yield an aqueous source of [I8F]fluoride for nucleophilic displacements while deuteron irradiation of neon will yield an anhydrous source of electrophilic fluorine-18 typically as elemental fluorine. However, as one reads on it will become apparent that many of these selection criteria are not as critical today as they were in the past. Now it is possible to render an aqueous source of [18F]fluoride anhydrous as well as interconvert the nucleophilic fluoride into electrophilic reagents. Electrophilic Fluorination Reagents Labeled with Fluorine-18 Electrophilic reagents create a chemical environment in which the fluorine atom is highly polarized with a positive charge. In this way, it is possible to fluorinate a variety of electron-rich substrates including alkenes, aromatic compounds and carbanions with fluorine-18. Over the years several reviews on the subject of electrophilic fluorination have been written. The reader is encouraged to seek out these works for greater detail on the subject (Kilbourn, 1990; Rozen, 1988; Wilkinson, 1992; Berridge, 1986). Without a doubt, electrophilic fluorination reactions are fast and efficient making them highly desirable synthetic pathways to achieve radiopharmaceuticals labeled with fluorine-18. The only downside seen is that most fluorine-18 labeled electrophilic reagents derive from [I8F]F2 which suffers from low specific activity. /.

Preparation of Fluorine-18 Labeled Elemental Fluorine:

The simplest reagent in this class of precursors is [18F]F2. It typically is produced through the deuteron bombardment of a high-pressure neon gas target containing 0.1 to 2% of carrier F2 (Casella et al., 1980). The method, however, suffers in specific activity due to the carrier addition with practical limits around 12 Ci/mmole (Blessing et al., 1986). Two variations on this approach involve proton irradiations of 18Oenriched O2 gas targets. Both methods rely on the fact that fluorine-18 will remain trapped on the target walls when there is no carrier present during the irradiation to chemically scrub the isotope. In the first approach, a mixture comprised of hydrogen and helium gases is swept through the target after bombardment while the target is heated to 600°C. The fluorine-18 is recovered as H[18F], and later converted to [I8F]F2 through microwave discharge using a mixture of 5% carrier F2 in helium. (Straatman et al., 1982) The rigors of heating a target to such high temperature eventually take their toll on pressure seals and internal surfaces. A second more practical variation on this concept involves the same target irradiations as described in the first with the exception that a small amount of carrier F2 is added to the target, and it is briefly re-irradiated. This manipulation allows for relatively efficient exchange of the fluorine-18 producing a source of [I8F]F2 (Nickels et al., 1984; Solin & Bergman, 1986). A more recent approach for generating higher specific activity [I8F]F2 (1.5 Ci/pmol) involves a two-step process beginning with [18F]fluoride produced from the 18O(p,n)18F reaction on 18O-enriched water (Bergman, 1997). The aqueous fluoride solution, as K18F, is mixed with kryptofix and acetonitrile followed by heating to dryness. A small amount of methyl iodide in acetonitrile solvent is then added to the dry residue to yield CH3[18F] within 1 minute. The CH3[|8F] is flushed with neon gas into a quartz discharge

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND

FLUORINE-18

251

chamber containing 150 nmole of carrier F2 where the mixture is discharged at 20-30 keV, 280 jiA, for 10 seconds resulting in about 30% conversion of the original [18F]fluoride to [18F]F2. ii. Preparation of Fluorine-18 Labeled Trifluoromethyl Hypofluorite: Over the years, several other electrophilic [18F]fluorinating agents have been successfully prepared and applied in the laboratory. [18F]Trifluoromethyl hypofluorite, CF3O[18F], was the first in a line of a subclass of "milder" electrophilic fluorinating agents that offered more regioselective control with less degradation to substrate (Neirinckx et a/., 1978). The process of production involves cesium fluoride mediated reaction between F2 and carbonyl fluoride. The reaction is conducted within an F2-passivated nickel irradiation target. Without carrier present, the target walls will retain all of the fluorine-18 radioactivity upon removal of the irradiation gas. The target is then used as a reaction vessel into which cesium, fluoride, F2 and carbonyl fluoride are introduced. Optimal results are obtained within 15 minutes of reaction at 110°C producing a 33% yield of CF3O[18F]. iii.

Preparation of Fluorine-18 Labeled Acetyl Hypofluorite:

The preparation of this important precursor has been re-examined over the years for a couple of reasons. Compared to [18F]F2, it is milder as a fluorinating agent. Perhaps more importantly, it possesses a much greater solubility over a wider range of reaction solvents. In the original methodology, [18F]F2 produced from a neon gas target is slowly emptied into a glass reaction vessel containing a solution of aqueous ammonium hydroxide in glacial acetic acid (Shiue etal., 1982).

[18F]F2

+

CH3CO2NH4

AceticAdd

»

CH3C0218F

+ NH 4 18 F

Reaction is almost immediate yielding 40% CH3CO2[18F] although the process of emptying the pressurized target in a controlled manner is a limiting factor. Most likely, any method for generating [18F]F2 will suffice for this reaction although deuteron irradiation on neon/F2 mixtures would seem the course to take. Preparation of this precursor was greatly simplified and made more reliable by the development of a gassolid phase reaction (Jewett et al., 1984; Chirakal et al., 1988).

[I8F]F2

+

AcOH'AcOK

*>

CH3CO218F

+

[18F]HF'AcOK

The method involves passing [18F]F2 through a column containing a complex of alkali metal acetate with acetic acid. The fluorine-18 reacts, and is retained on the column as CH3CO218F, which can be removed in an aqueous rinse. iv.

Preparation of Fluorine-18 Labeled Perchloryl Fluoride:

Perchlorofluoride, FC1O3, as an electrophilic reagent will react with unfunctionalized aryllithium compounds, such as phenyl lithium, to produce modest yields of the respective aryl fluorides (Muchowski & Venuti, 1980). Even so, its general utility as a fluorine-18 labeling agent has never really been fully exploited.

HANDBOOK OF RADIOPHARMACEUTICALS

252

Examples where it has been successfully used include the syntheses of [18F]-labeled 2-fluoroaniline, 2fluoroanisole, arid 3-fluoroveratrole in modest yields. (Ehrenkaufer et a/., 1983a) OCH3

OCH,

OCH3 18

OCH3 FC103

(24%)

The method for producing [18F]C1O3 involves passing [I8F]F2 through a column containing KC1O3 maintained at 90°C.

[18F]F2

KClO3

18

FClO3

K18F

Rapid on-line purification of the precursor is achieved by passing the effluent from the reaction column through a series of two solid-phase scrubbers containing granular NaOH and Na2S2O3. These scrubbers must be large enough to effectively remove any unreacted F2 and chlorinated oxides that may form in the KClO3 reaction. Although the reaction is quantitative, only half the radioactivity ends up as [18F]ClO3 with the remainder consumed as K[18F]. v.

Preparation of Fluorine-18 Labeled Xenon Difluoride:

Like the preceding precursors, [18F]xenon difluoride, [18F]XeF2, has received only limited attention where its efficacy for synthesizing [18F]-2-fluoro-2-deoxy-D-glucose and L-[18F]6-fluorodopa has been demonstrated (Sood et al., 1983; Firnau et al., 1980). The precursor can be prepared through a couple of approaches. The most common involves the thermal reaction between [18F]F2 and xenon gas in a sealed nickel vessel maintained at 390°C.

253

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-I8 18

[F]F 2

+

[18F]XeF2

Xe

The reaction typically takes 40 minutes to achieve a 70% yield. However, due to radioactive decay during this reaction time a specific activity of only about 450 mCi/mmol is achievable. (Chirakal et al., 1984) A second method involves isotopic exchange between H[18F], or some similarly suitable Bronsted or Lewis acid such as [18F]SiF4 or [!8F]AsF5, and XeF2. The reaction involves treating sulfuryl chloride solutions of XeF2 with the radiolabeled acid in fluorinated ethylene propylene vessels (Schrobilgen et al., 1981). Yields are typically low (<30%) and erratic, and precursor specific activity is low. A simpler approach involves [18F]fluoride ion exhange reaction with XeF2 that is catalyzed by the Cs+-Kryptofix 2.2.2. complex (Constantinou et a/., 2001). The complex acts to ionize the XeF2 when the reaction is performed in chlorinated solvents such as methylene chloride or chloroform. The catalyzed exchange reaction is much more efficient producing on average 60% yields of [18F]XeF2 from 50 minutes of reaction at room temperature, but doesn't provide any advantage in specific activity as approximately 50 mg of XeF2 is needed in the exhange.

18 -

F

XeF2

Cs — Krypotofix 222 CH2C12 20° C

[18F]XeF2

19,

vi.

Preparation of Fluorine-18 Labeled N-Fluoropyridinium Triflate: N-Fluoropyridinium salts have also been investigated as potential fluorine-18 radiolabeling agents (Oberdorfer et a/., 1988a). [18F]-N-Fluoropyridinium triflate was the first, and only, of a potential series of analogous N-fluoro-compounds that was tested. It can be readily prepared by direct reaction between [18F]F2 and N-trimethylsilylpyridinium triflate in acetonitrile solvent at -42°C yielding 46% of the radiolabeled precursor with specific activity of 167 mCi/mol.

[18F]F2 OSO2CF3 Si(CH3)3

CH3CN

18

FSi(CH3)3

-42°C

N

- OSO2CF3

18

The precursor exhibits high efficiency for reacting with Grignard compounds, related carbanions, and enolates yielding the corresponding [18F]-labeled products in high yields.

HANDBOOK OF RADIOPHARMACEUTICALS

254

THF

CH3(CH2)6MgCl

N

OSO2CF3

CH (CH2)6

ether

18

18,

(78%)

OCH3

CH2C12

OCH.

vii.

Preparation of Fluorine-18 Labeled l-Fluoro-2-Pyridone:

18

l-[ F]Huoro-2-pyridone as an electrophilic fluorinating agent also exhibits excellent qualities in terms of its chemical reactivity to undergo efficient

18

F-for-metal exchange with organometallic compounds. (Oberdorfer et al., 1988b) For

I8

example, reaction of 1-[ F]fluoro-2-pyridone with methyl lithium will yield CH3[18F] quantitatively.

[18F]F2 OSi(CH3)3

+ CH3Li

18

CFCl3

ether

FSi(CH3)3

*• CH318 F

O 18,

Carrier added l-[18F]fluoro-2-pyridone can easily be prepared in yields of 40-49% (out of a maximum possible yield of 50%) by bubbling [18F]F2 through a solution of 2-(trimethylsiloxy)pyridine in CFC13, at low temperature. Typically, the process takes about 1 hour which includes the time to irradiate and deliver [18F]F2 to the reaction vessel. The pure precursor is obtained by subliming the crude reaction residue for 30 minutes.

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND viii.

Preparation of Fluorine-18 Labeled

FLUORINE-18

255

N-Fluoro-N-Alkylsulfonamides:

The alkylsulfonamides represents yet another line of "mild" radiofluorinating agents that were developed to regiospecifically react with a variety of carbanions and organometallic compounds (Satyamurthy et al., 1990a), As with the preceding precursors, the sulfonamides are readily labeled with fluorine-18 by bubbling [18F]F2 through a solution of the appropriate sulfonamide in Freon at -78°C. Reaction is almost immediate, and the solvent easily removed through evaporation at room temperature. The reaction residue can be taken up in some suitable solvent like ether for subsequent reaction. Typically, 45% radiochemical yields of the radiolabeled sulfonamide can be obtained, with subsequent reaction carried out in the same vessel in which the precursor was prepared.

[18F]F2

CH3 M= Li, MgBr On testing reactivity of various sulfonamides, endonorbornyl-p-tolylsulfonamide exhibited the highest level toward exchange yielding [l8F]fluorobenzene in roughly 60% yield. Nucleophilic Fluorinating Agents Labeled with Fluorine-18 i.

Preparation of Fluorine-18 Labeled Fluoride Ion:

Today, radiofluorinations based on nucleophilic processes rely almost exclusively on no-carrier-added [I8F]fluoride as the labeling precursor. For years, a number of problems which greatly affected [18F]fluoride reactivity had to be solved in order for radiochemists to successfully move ahead with this precursor. Several papers discuss these issues (Kilbourn, 1990; Brodack et al,

1986; Gatley et al, 1986; Gatley and

Shaughnessy, 1981; Kilbourn et al., 1986; Coenen, 1989). One key issue is how to render the [18F]fluoride in a suitable solvent that is devoid of an excess of water. Since the method of choice for producing no-carrier-added fluorine-18 is the

18

O(p,n)18F reaction on

18

O-

enriched water targets, it is necessary that the fluoride and aqueous media be separated prior to subsequent chemistry. It is worth mentioning that a cryogenic target design relying on a frozen state of

!8

O-enriched

carbon dioxide during irradiation generates reasonable yields of fluorine-18, and provides an easy way to separate the target material from the radioisotope after bombardment (Firouzbakht, et al., 1999b).

256

HANDBOOK OF RADIOPHARMACEUTICALS

Essentially, the target material is thawed and the enriched gas recovered leaving the fluorine-18 deposited on the walls. However, even in this instance, the [l8F]fluoride was recovered through basic aqueous rinses of the target bringing us back to the same issue of anhydrous fluoride. Typically, most nucleophilic radiofluorinations will tolerate trace levels of water in the reaction medium so it is not essential, and probably next too impossible to render the [18F]fluoride entirely anhydrous. Simple distillation of the aqueous phase can lead to two problems. On the one hand, recovery of the enriched water while maintaining isotopic purity may not be effective. More importantly, distillation can lead to a concentration of anionic and cationic contaminants from the target materials that can influence reactivity of the [18F]fluoride (Nickles et al., 1986). Cations, especially A13+ and Ca2+, will probably impinge on reactivity the most. A number of procedures have been described for isolating [18F]fluoride and target water that render it in suitable reaction media. Typically, the target water is processed through an ion exchange resin that provides a means not only to recover the target water, but also a way to remove some of the target water ion contaminants that can impact on reactivity. One way involves using Dowex AG1-X8 anion exchange resin (Schlyer et al., 1987; Jewett et al., 1988; Schlyer et al., 1990). Interestingly enough, this resin seems to have a high affinity for metal cations, as well. Extensions of this approach have also been described. One includes using quanternary ammonium resins for isolation of the [18F]fluoride from the target water, as well as for creating a reactive nucleophilic media for subsequent chemistry (Mulholland et al., 1988; Mulholland et al., 1989). Additionally, a supported aminopolyether can be used to a similar extent as the quanternary ammonium resin with perhaps some enhancement of fluoride reactivity (Hamacher et al., 1990). Finally, [18F]fluoride can be extracted from target water using a potassium ion/cryptand complex immobilized on a stationary support (Jewett et al., 1988). Two other approaches are worth mentioning because they don't rely on a trapping agent to achieve separation of the radioisotope and target water. One method utilizes electrochemical deposition as a means to achieve reasonable extraction efficiency of [18F]fluoride from target water (Alexoff et al., 1989). By controlling the polarity on an applied potential across an electrochemical cell, it is possible to selectively deposit [18F]fluoride on the cell's walls and later remove it with reasonable efficiency. Unfortunately, the technique has not found wide-spread acceptance. The other method involves chemically converting the [18F]fluoride into gaseous [18F]fluorotrimethylsilane to achieve separation. The gas can be trapped in near anhydrous acetonitrile and hydrolyzed back to fluoride using a small amount of base(Gatley, 1989). Another key issue is how to maintain [18F]fluoride solubility. Without a doubt this is the most important requirement for successful nucleophilic radiofluorinations. A counterion is usually required that possesses sufficient solubility within the reaction media to maintain fluoride solubility, as well. [l8F]fluoride is extracted from the anion exchange resin using a dilute alkali metal carbonate solution. Typically, potassium carbonate is preferred. However, the K+ counterion possesses limited solubility in some reaction solvents. Larger alkali metals such as cesium or rubidium do offer some enhancement to fluoride solubility although they are still limited in some respects. Even so, these salts have been successfully used in a number of radiofluorinations (Shiue et al., 1985; Shiue et al., 1986a; Shiue et al., 1986b).

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND

FLUORINE-18

257

A number of alternate methodologies have been investigated to provide enhancement to solubility, as well as to reactivity. Some of these involve adding complexing agents to enhance cation solubility, while others explore different cations altogether. For example, addition of 18-crown-6 ether will greatly improve [18F]fluoride reactivity in certain instances such as radiofluorinating progesterone (Me et al., 1982; Irie et al., 1984). Use of aminopolyether Kryptofix 2.2.2. as a complexing agent will also improve K+ solubility, and greatly enhance nucleophilic radiofluorinations with [18F]fluoride on both aliphatic and aromatic substrates. Examples of successful radiofluorinations include the preparation of [18F]-2-deoxy-D-mannose, [18F]-2-deoxy-D-glucose, [18F]-N-methylspiperone,

[18F]-spiperone, as well as [18F]-aliphatic carboxylic

acids (Block et al., 1986; Coenen, et al., 1986a; Hamacher et al., 1986a; Hamacher et al., 1986b; Hamacher et al., 1986c). A good choice for alternate cations includes the tetraalkylammonium salts, R4N+ (R=methyl, ethyl or butyl). These salts are extremely proficient at promoting nucleophilic radiofluorinations without the need for additional complexing agents. They also offer greater utility in terms of their ability to remain solubilize in a variety of solvent classes (Kiesewetter et al., 1986). ii. Preparation of Fluorine-18 Labeled Hydrogen Fluoride: Fluorine-18 labeled hydrogen fluoride, H[18F], can be used in nucleophilic radiofluorinations of aromatic compounds by the Schiemann or triazene decomposition reactions although the former requires the presence of carrier, and neither reaction is terribly efficient (De Kleijn, 1977; Ng et al., 1981; Barrio et al, 1983; Berridge et al., 1985; Satyamurthy et al., 1990b). Shortly after the development of the neon gas target for [18F]F2 production using the 20Ne(d,a)18F nuclear reaction, researchers quickly realized that in the absence of any reactive scavenging gas, the fluorine-18 remains trapped to the inside walls of the target. This phenomenon can be exploited as a way to prepare large quantities of presumably anhydrous H[18F] for nucleophilic radiofluorinations. This process, however, involves heating the target after irradiation up to 1000°C while flushing hydrogen gas through it (Winchell, 1976). The hydrogen gas reacts with the surface bound fluorine-18, and allows it to be harvested as H[18F]. A similar strategy can be applied to irradiations of oxygen-18 enriched O2gas. Unfortunately, the rigors of heating a metal target to such extreme temperatures will eventually take their toll on target surface morphology, as well as on target hardware. A more practical approach involves the addition of hydrogen gas to the target during the irradiation. Non-heated recirculating gas targets work, but only generate modest amounts of H[I8F] (20-30 rnCi) (Tewson & Welch, 1980; Levy et al., 1982). By combining the features of heating the target during the irradiation along with adding hydrogen gas to the neon will produce much larger amounts of H[18F] (Blessing et al., 1986; Clark & Buckingham, 1982; Ehrenkaufer et al, 1983b; Kilbourn et al., 1982). The advantage here is that the target doesn't have to be heated to such extreme temperatures as in the post-irradiation treatment described above. Even so, this approach is not terribly dependable for consistent recovery of no-carrier-added H[18F]. Improved reliability can be achieved, at the cost of specific activity, through the addition of small amounts of carrier HF. Even so, controlling the amount of carrier introduced is not trivial. Anhydrous HF gas is highly corrosive requiring suitable valves and plumbing to safely manipulate small amounts of the material. A variation on this strategy uses mixtures of CF4 and H2 in neon. Small but adequate amounts of carrier HF are generated in situ presumably through radiolysis (Ferrieri eraL, 1982).

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HANDBOOK OF RADIOPHARMACEUTICALS

Preparation of Fluorine-18 Labeled Alkylating Agents Direct nucleophilic substitution with no-carrier-added [18F]fluoride is often difficult and sometimes even impossible to carry out in certain complex molecules. A classic example is the aryl radiofluorination of phenolic compounds. Due to the acidity of the phenolic hydrogen, abstraction of hydrogen by fluoride will dominate over substitution. Researchers quickly realized that there was a need to expand the arsenal of radiofluorinating agents beyond the scope of the simple electrophilic precursors, and "naked" fluoride. An alternate labeling strategy evolved for introducing fluorine-18 onto larger molecules by first attaching the radioisotope to a prosthetic group. One of the first areas to be developed involved the preparation and application of bifunctional [18F]fluoroalkanes. The intent here was to replace radiopharmaceuticals labeled with [11C]alkyl iodides with near equivalent compounds labeled with longer-lived fluorine-18. 18

(CH2)nX F

+

n= 1– 3 X=Cl, Br, I

R-Z-H

*•

18

R-Z-CH218 F

+

XH

Z=functional groups containing N, O, S or C

No-carrier-added CH2Br[18F] was the first to be prepared in this class of precursor (Coenen et al., 1986b). It can be readily prepared by exchange of l8F-for-Br through nucleophilic substitution of [l8F]fluoride with CH2Br2. Typically, the reaction is carried out in anhydrous acetonitrile at 115°C, and the volatile radiolabeled product collected cryogenically using liquid nitrogen. The trick to getting an efficient reaction here is to render the fluorine-18 in a reactive form. As described earlier, this is readily accomplished by adding potassium carbonate and aminopolyether Kryptofix 2.2.2. as a complexing agent to the aqueous phase containing the fluorine-18. Yields of 62% can be attained in this fashion. More recently [18F]fluoromethyl iodide, CH2I[18F], was prepared using much the same strategy as described above where reactive [18F]fluoride is mixed with diiodomethane (Zheng & Berridge, 2000). Although yields are not as high (40% in 15 minutes time) as the bromide form, an advantage here is that cryogenic trapping of the precursor does not have to be as rigorous. A dry ice bath at -20°C will perform nicely. A number of applications for labeling with CH2I[18F] have also been tested and it seems that the iodo form is slightly more reactive toward SN2 reactions.

259

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

(60%)

O II

18

C-OCH2 F (57%)

18

CH2SH

CH2SCH218F (12%)

ONa 18

This class of precursor was quickly expanded to include [18F]fluoroalkanes with larger carbon side-chains. However, the size of the alkyl side-chain, as well as the nature of the leaving group, can strongly influence the nucleophilic substitution yield where higher yields are usually obtained for larger side-chains, and for a sequence of leaving groups Br < mesyl < tosyl (Block et al., 1987). The choice of starting material for larger side-chains is somewhat independent of the leaving substituent, and can be made on the basis of commercial availability. It should be noted, however, that symmetrical bistosyloxyalkanes offer a greater advantage with respect to the stability of the educts and the fluorinated products when compared to the respective halides. Even so, the number of successful applications of N-alkylation of neurotransmitter receptor active amides and amines using [18F]fluoroalkyl halides suggests that these precursors can work equally well in many circumstances (Shiue et al., 1987). Examples below illustrate that radiolabeling of N-(3-[18F]fluoropropyl)lorazepam and N-(2-[18F]fluoroethyl) and N-(2-[18F]fluoroproyl) spiroperidols by this approach can be accomplished with very good yields.

HANDBOOK OF RADIOPHARMACEUTICALS

260

(CH2)318F

I(CH2)318F

I(CH2)n18F (n=2,3)

(50%)

Successful radiofluorination of H-acidic compounds can also be carried out using [l8F]fluoroalkylating agents (Block et al., 1988a). As mentioned earlier, compounds in this class, such as the phenols, can not be radiofluorinated with "naked" fluoride. However, it is possible to accomplish this task in high yields using suitable [18F]fluoroalkylating agents. In this instance, the best no-carrier-added labeling yields are obtained using tosylates as leaving groups.

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18 18

261

aminopolyether

FCH2CH2OTos

OH

18

222 Kryptofix/K+

(56%) The trifluoromethylsulfonates (triflates) are also good leaving groups for inducing efficient nucleophilic exchange with [18F]fluoride to yield the respective [18F]alkylating agent. Both 3-bromopropyl-l-triflate and 3-iodopropyl-l-triflate can be used as starting materials for preparing useful amounts of the labeling precursors, 3-[18F]fluoropropyl bromide and 3-[18F]fluoropropyl iodide. Their usefulness in radiofluorinations has been demonstrated in the labeling of several diprenorphine derivatives, (Chesis & Welch, 1990) of certain dopamine D-l and benzodiazepine receptor radioligands, (Teng et al., 1990) as well as in the labeling of certain derivatives of spiperone (Oh et al., 1999).

Preparation of Fluorine-18 Labeled Acylating Agents Developments in radiolabeled peptides and antibody fragments possessing relatively fast in vivo kinetics for receptor and immunoimaging quickly lead to a need for ways to radiofluorinate large biologically active molecules at no-carrier-added levels. While radiofluorination of peptides by electrophilic fluorination has been shown to proceed with reasonable efficiency, the process requires carrier fluorine which is not tolerable in many instances (Hebel et al., 1990). Chemists turned their attention to developing a new class of labeling precursor based on no-carrier-added [18F]fluoroacylating agents. Agents in this class include [18F]fluorocarboxylic acids, their esters and the acid halides. In the early stages of development, fluoroacylation with no-carrier-added 2-[18F]fluoropropionic acid methylester was successfully applied to primary alcohols and amines (Block et al., 1988b). C2H5OH

H H3C—C—COOC2H5

H I H3C-C— COOCH3 18

+

CH3OH

18

J

H

H2N(CH2)3CH3

H3C-C-CONH(CH2)3CH3 18

This synthetic scheme can be optimized for reactions with amines by incorporating an additional step to convert the ester to its free acid form using base mixed with dicyclohexylcarbodiimide.

262

HANDBOOK OF RADIOPHARMACEUTICALS H I

_

OH

H |

H3C-C— COOCH3 - *• H3C-C-COO F

H O H

_ dicyclohexyl caibodimide

|

||

|

>> H3C-C— C-N-R -

F

F

The preparation of 2-[18F]fluoropropionic acid methylester is similar to what has already been described for the [18F]fluoroalkylating agents. Activation by the aminopolyether 2.2.2/K2CO3 complex is used for the nucleophilic fluorine-18 exchange in a-substituted acid esters.

Increasing yields for formation of the

precursor are found with the sequence of leaving groups: I<
Fluorine-18 Labeled Fluoroaryl Precursors A number of no-carrier-added fluorine-18 labeled aromatic compounds can be made that add enormous versatility in the chemist's arsenal of labeling synthons to allow him to tackle rather complex mult-step syntheses of radiopharmaceuticals successfully. No-carrier-added synthesis of many of these key intermediates usually involves nucleophilic aromatic substitution. This process most often proceeds with high efficiency, and in high yield for radiofluorinations involving [18F]fluoride ion displacement of either a nitro group, halogen atom or a N+(CH3)3 group on an aromatic ring that is activated by some strongly electronwithdrawing function such as a cyano, nitro or keto group (Angelini et al., 1985; Attina et al., 1983; Shiue et al., 1984). It has been possible to use this reaction in the presence of electron-donating groups (Ding et al., 1990).

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

263

i. Preparation of [!8F]fluorobenzaldehydes: [ F]Fluorobenzaldehydes are regarded as extremely useful precursors not only for their ability to produce other radiofluorinating agents such as [18F]benzyl alcohols and [18F]benzyl halides, but also because they can be used directly in the preparation of some rather complex radiopharmaceuticals. 4-[18F]Fluorobenzaldehyde was first successfully used for the synthesis of the amino acid D,L-4-[18F]fluoroalanine (Lemaire et al., 1987). Other examples using either 4-[18F]fluorobenzaldehyde or 6-[18F]fluoropiperonal include the radiolabeling of L-6-[18F]fluorodopa (Lemaire et al., 1990), L-4-[18F]fluorotyrosine (Lemaire et al, 1991), the MAO inhibitor [18F]fluorodeprenyl (Plenevaux et al., 1991), as well as the false adrenergic transmitter [18F]fluoronoreprinephrine (Ding et al., 1991). Reductive amination of these fluoroaidehydes in the presence of suitable secondary amines can also be a good source of radiopharmaceuticals possessing a benzyl amino group. [18F]Fluorotropapride, a D2 antagonist, is prepared in this manner (Damhaut et al., 1991). Oxidation of [18F]fluorobenzaldehydes by the Baeyer-Villiger reaction followed by dealkylation using boron tribromide will also produce [18F]fluorocatechol (Chakraborty & Kilbourn, 1991). 18

[18F]Fluorobenzaldehyde is typically prepared in high yield by radiofluorination of o- or p-nitrobenzaldehyde using [18F]fluoride activated with Kryptofix 2.2.2./K+ as a complexing agent (Lemaire et al., 1987; Lemaire etal., 1992).

[K/Kryptofix 2 2 2 ] 1 8 ] DMSO, 140ºC NO

(50%) The reaction is typically carried out in 1 mL DMSO solvent using about 15 mg of the corresponding nitroaldehyde. Reaction time is 20 minutes with heating in a closed vessel at 130–140°C. The crude product is purified using a C-18 Sep Pak to extract the product and tetrahydrofuran or some suitable solvent to eventually extract the aldehyde for subsequent reaction. Radiofluorination yields in this step are usually >50%. ii. Preparation of [18F]fluorobenzylalcohols: The [18F]fluorobenzylalcohols are important only in the sense that they are key intermediates to generating [18F]fluorobenzylhalides. These compounds are prepared from their respective [18F]fluorobenzaldehydes using the procedures described above. However, purification of the radiofluorinated aldehyde is not necessary after the first step of the reaction as reduction to the alcohol can be carried out in the same vessel. The contents must be cooled, however, at the end of the first step after which 10 mg NaBH3CN and a trace of bromocresol green are added. The mixture must also be made acidic (pH 4) typically by adding dilute HCl/methanol (Hatano et al., 1991). Reduction using lithium aluminum hydride is also possible, but the added complexity to render the [18F]fluorobenzaldehyde anhydrous, as well as devoid of DMSO creates

HANDBOOK OF RADIOPHARMACEUTICALS

264

much more complexity than is needed. A third approach is appealing because of its simplicity. The method uses solid-phase reduction involving a small column of NaBH4 supported on aluminum oxide (Lemaire et al., 1991). The reaction contents from the [18F]fIuorobenzaldehyde synthesis are simply passed through a potassium carbonate drying tube before entering the reduction column. The [18F]fluorobenzyl alcohol is directly recovered from the column using THF solvent.

NaBH4 • A12O3

iii.

Preparation of [18 F]fluorobenzylhalides:

Within the class of [18F]fluoroaryl precurors, the [18F]fluorobenzylhalides are perhaps the most versatile in the sense that they provide a widest range of useful radiopharmaceuticals by simple [18F]fluorbenzylation. The usefulness of [18F]fluorobenzylations in radiopharmaceutical synthesis was first demonstrated with the preparation of several radiolabeled benzamide neuroleptics that exhibited high specific binding toward D2dopamine receptors in vivo (Hatano et al., 1991). In addition, several other receptor ligands have been prepared by this approach including 2- and 4-[18F]fluorotropaprides (Damhauat et al., 1992), [18F]NCQ 115 (Halldin et al, 1994), andp-[18F]fluorobenzyltrozamicol (Efange et al., 1994). [18F]Fluorobenzyl halides can be prepared in a number of ways, most of them involving complicated multistep reactions. All begin with the preparation of a [18F]fluorobenzaldehyde using the methods described above. From this point one of four ways can be used to convert the fluoroaldehyde into the appropriate fluorobenzyl halide. The first three methods require a reduction step to convert the aldehyde to the corresponding alcohol. The best ways to accomplish this task have already been described in the preceding section under [18F]fluorobenzyl alcohols. In the first method, which was tailored for making 4[18F]fluorobenzyl iodide, the [18F]fluorobenzaldehyde is reduced to the alcohol using a solution of lithium aluminum hydride in THF solvent (Mach et al., 1993). As mentioned earlier, care must be taken to remove excess water and DMSO solvent from the proceeding reaction. Reduction occurs rapidly and efficiently at room temperature with adequate stirring, after which the solvent is evaporated. The radiofluorinated alcohol remains complexed as the lithium salt. Hydrolysis of the salt and iodination of the free alcohol is accomplished using hydroiodic acid (57%) and reaction for 3 minutes at 90°C. This can be carried out in the same reaction vessel as that used in the aldehyde reduction. After the iodination reaction, the crude product is purified through extraction with a C-18 Sep Pak. A variation on this approach uses hydrobromic acid, HBr, in place of the hydroiodic acid to make [I8F]fluorobenzyl bromide (Hatano et al., 1991).

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

LiAfflL

HX

THF

90° C

265

(50%) In the second method, either thionyl chloride or thionyl bromide can be used in place of hydroiodic or hydrobromic acids to convert the alcohol to the corresponding halide (Hwang et al., 1991; Damhaut et al., 1992). This method offers a slight advantage over the previous one in that the THF solvent used either in the reduction step or in the [18F]fluorobenzyl alcohol extraction does not have to be evaporated to dryness. Volumes are reduced slightly and reaction carried out for 1-2 minutes at 110°C in a sealed vessel. Yields on average are slightly less than the previous method, but certainly adequate.

NaBH4 THF

SOX, 110°C

A third method involves direct reductive iodination of [18F]fluorobenzaldehyde using diiodosilane, (Lemaire et al., 1994; Dence et al., 1997). While the method does eliminate the need for an intermediate reduction step, one must still isolate the aldehyde from the DMSO solvent prior to the iodination step.

SiH2I2

The last method offers one key advantage over the other methods in that the processes are greatly simplified by utilizing a solid-phase reaction in the reduction step followed by halogenation using dihalotriphenylphosphine, PH3PX2 (X=Br, I). The entire process is amenable to system automation requiring about 30 minutes (Iwata et al., 2000). Once the [18F]fluorobenzaldehyde is made it is reduced to the alcohol using a solid-phase reaction involving alumina supported NaBH4. This procedure was described earlier. The halogenation step is carried out at room temperature in CH2Cl2 using about l0mg of either PH3PBr2 or PH3PI2- Conversions of

266

HANDBOOK OF RADIOPHARMACEUTICALS

the alcohol to the bromide are near quantitative for 2 minutes of reaction, and somewhat less to convert to the iodide.

Ph3PX2

NaBH

iii.

l8

Preparation of [[18F]fluoroalkylbenzylsulfonate

esters:

An extension of the [18F]benzylating agents includes the small group of [18F]fluoroalkylbenzylsulfonate esters which provide a way to attach radiofluorinated aromatic groups with longer carbon side-chains. (Choe et al., 1998) The method for making these precursors involves [18F]fluoride displacement of the corresponding bisulfonate ester. The incorporation of [l8F]fluoride into 1,4-benzenedimethanol bimesylate or bitosylate is somewhat low (on average 32%), but acceptable for subsequent radiolabeling. In application, the [18F]fluoromethylbenzylsulfonate ester was prepared and reacted with spiperone and 1 -phenylpiperizine to yield 3-N-(4-[18F]fluoromethylbenzyl)spiperone and l-N-(4-[18F]fluoromethylbenzyl)-4-phenylpiperazine as products demonstrating the precuror's efficacy for radiolabeling amides and secondary amines.

_18

f FJFluoride n-Bu4NOH, THF 5 min., 90ºC (X=Mes, Tos)

XO

XO

iv. Preparation of 18, [18FJfluorohalobenzenes: [18F]Fluorohalobenzenes are useful precursors in the synthesis of a variety of fluorine-18 labeled aryllithium, aryl-magnesium and aryl-zinc compounds, which can be used to form carbon-carbon bonds through their reaction with suitable electrophilic reagents. A number of methods exist for producing this class of precursor although only two are acceptable for radiofluorination of receptor-based imaging agents. The first reported method involved recoil labeling of halobenzenes using a target comprised of an appropriate halobenzene mixed with hexafluorobenzene (Berei et al., 1974; Berei et al., 1987). Ruorine-18 was produced using the 18

19

F(n,2n)18F nuclear reaction.

As is typical with this approach, radiochemical yields of the

[ F]fluorohalobenzene are <1% with low specific activity.

SYNTHETIC PRECURSORS LABELED WITH CARBON-11 AND FLUORINE-18

267

A second approach relies on electrophilic fluorination using [18F]F2 or [18F]acetyl hypofluorite on parasubstituted phenyl derivatives of tin or other suitable metal (Coenen et al., 1987). While radiochemical yields are typically in excess of 60%, specific activity of the final product is usually unsuitable to make this approach practical. A third approach involves nucleophilic substitution of no-carrier-added [18F]fluoride producing 4[18F]fluorochlorobenzene in a 14% yield after a rather long (85 minute), and complicated three-step synthesis (Feliu, 1988). The process involves [18F]fluoride exchange on 1,4-dinitrobenzene followed by reduction and diazotation to yield [l8F]-p-fluorobenzene-diazonium chloride. The key reaction in this three-step synthesis is the reduction of the diazonium chloride using sodium cyanoborohydride. The final approach is very attractive because it requires only a single step involving direct nucleophilic aromatic substitution of [18F]fluoride on appropriate halophenyl-trimethylammonium salts. The method was applied to the preparation of 4-[18F]fluorobromobenzene and 4-[18F]fluoroiodobenzene in relatively high yield (50%).

(CH3)3N + .18

[18F]Fluoride K + /Kryptofix222

Q

X

X

(X=Br, I) (An =TfO - , I-, Tos O-, MeOSO2O-)

v.

Preparation of [18F]fluoroarylketones:

Attempts to expand the scope of radiofluorinations of large biologically active molecules resulted in the development of p-[18F]fluorophenacyl bromide as a radiofluorinating agent (Kilbourn et al., 1987; Downer et al., 1997). In additon to providing a means to radiofluorinate amine sites of large molecule, p[18F]fluorophenacyl bromide can be potentially useful for alkylating the thiol of free cysteine to form the corresponding thioether, or the methionine residues of proteins (Glasel et al., 1966; Kanstrup et al., 1993). p-[18F]Fluorophenacyl bromide can be prepared by two methods both relying on the intermediate synthesis of p-[18F]fluoroacetophenone (Dence et al., 1993). The p-[18F]fluoroacetophenone is prepared by nucleophilic substitution of [18F]fluoride ion for the nitro group on p-nitroacetophenone. Typically, 55–60% radiochemical yields are obtained in 5 minutes of reaction using microwave heating of a sealed 5 mL Reactivial™ containing 2 mg of substrate dissolved in DMSO (Hwang et al., 1987). After reaction, the p-[18F]fluoroacetophenone is purified using a C-18 Sep Pak. The first method for converting the radiolabeled acetophenone to p[l8F]fluorophenacyl bromide involves dissolving the intermdiate in THF containing a small amount (600 mg)

HANDBOOK OF RADIOPHARMACEUTICALS

268

of Perbromide on Amberlyst™ A-26 resin. After 10 minutes of reaction at 60°C, the mixture can be easily extracted with dilute thiosulfate solution, and purified by Sep-Pak to generate 65% yield of the radiofluorinating precursor.

18

F ,DMSO

microwave 5 min.

A-26 Br3- , THF

60°C, 10 min.

NO2 In a second less efficient method, the radiolabeled acetophenone is mixed in glacial acetic acid treated with 10 mg of 4-(dimethylamino)pyridiniumbromide perbromide and heated for 10 minutes at 90°C. Final extraction generates about 42% of purified precursor, somewhat lower than the resin approach, but acceptable. Conclusions It is evident from this chapter that there is enormous flexibility both in the selection of the nature of the radioisotope and ways to generate it, as well as in the selection of the labeling precursor to appropriately attach that radioisotope to some larger biomolecule of interest. The arsenal of radiolabeling precursors now available to the chemist is quite extensive, and without a doubt will continue to grow as chemists develop new ones. However, the upcoming years will perhaps reflect a greater effort in refining existing methods for preparing some of those precursors that are already available to us. For example, the use of solid-phase reactions to accomplish in a single step what would normally take several using conventional solvent-based reactions has already been shown to work in many occasions. The obvious advantage here is that processes become more amenable to system automation thus affording greater reliability in day-to-day operations. There are perhaps other technologies in science that have yet to be realized by the chemist in the PET laboratory that could provide a similar or even a greater benefit. One only needs to be open to new ideas, and imaginative enough to apply them to the problems at hand. Acknowledgements This research was carried out at Brookhaven National Laboratory under contract DE-AO02-98CH10886 and with the U.S. Department of Energy and supported by its Office of Biological and Environmental Research, and also by the National Institutes of Health (National Institute on Drug Abuse and National Institutes of Neurological Diseases and Stroke).

SYNTHETIC PRECURSORS LABELED WITH CARBON-! 1 AND FLUORINE-IS

269

REFERENCES Aamodt RL, Peterson V and Phillips R (1952) 12C(p,pn)11C cross section from threshold to 340 MeV. Phys. Rev., 88, 739–742. Alexoff D, Schlyer DJ and Wolf AP (1989) Recovery of [18F]fluoride from [18O]water in an electrochemical cell. Appl. Radiat, hot. 40, 1–4. Andersson Y and Langstrom B (1994) Transition-mediated reactions using [11C]cyanide in synthesis of 11Clabeled aromatic compounds. J. Chem. Soc., Perkin Trans., 1,1395–1400. Angelini G, Speranza M., Wolf AP and Shiue C -Y (1985) Nucleophilic aromatic substitution of activated cationic groups by 18F-labeled fluoride. A useful route to no-carrier-added I8F-labeled aryl fluorides. J. Fluorine Chem. 27, 177–187. Antoni G, Malmborg P and Langstrom B (1989) Synthesis of11C-labeled halonitriles and examples of their use in alkylation reactions. Ann. Univ. Turkuensis, D27, Proceedings from the 5th Symposium on the Medical Applications of Cyclotrons, Turku Finland. Antoni G, Malmborg P and Langstrom B (1991) Synthesis of [CN-11C]acrylonitrile. J. Label Cmpds. Radiopharm. 30, 148,Proceedings of the 8th Int. Symposium on Radiopharmaceutical Chemistry, Princeton, USA. Attina M, Cacace F and Wolf AP (1983) Displacement of a nitro group by [18F]fluoride ion. A new route to aryl fluorides of high specific activity. J. Chem. Soc., Chem Commun. 108. Backhausen H, Stocklin G and Weinreich R (1981) Formation of Aero., 29, 1–5.

18

F via its 18Ne precursor. Radiochim.

Balatoni J, Adam M and Hall L (1989) Synthesis of 11C-labeled aromatics using aryl chromium tricarbonyl intermediates. J. Label. Cmpd. Radiopharm. 27, 1429–1435. Barrio JR, Satymurthy N, Ku H and Phelps ME (1983) The acid decomposition of l-aryl-3,3-diakyltriazenes. Mechanistic changes as a function of aromatic subtitution, nucleophilic strength and solvent. J. Chem. Soc. Chem. Commun., 443–444. Berei K, Vasaros L, Noreeyev YV and Sutyor J (1987) Substitution effects in hot replacement by recoil halogens. Radiochim. Acta. 42, 13–20. Berei K and Vasaros L (1974) Reactions of recoil fluorine in liquid halobenzenes. Radiochim Act., 21, 75–82. Berger G, Maziere M, Prenant C and Comar D (1980) Synthesis of carbon-11 labeled acetone. J. Appl. Radiat. hot., 31, 577–578. Bergman J and Solin O (1997) Fluorine-18-labeled fluorine gas for synthesis of tracer molecules. Nucl. Med. & Biol, 24, 677–683. Berridge MS, Crouzel C and Comar D (1985) Aromatic fluorination with NCA F-18 fluoride: a comparative study. J. Label. Cmpds Radiopharm. 22, 687-690. Berridge MS and Tewson TJ (1986) Chemistry of 18F radiopharmaceutical. Appl. Radiat. Isot., 37, 685–694.

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Satyamurthy N, Barrio JR, Schmidt DG, Kammerer C, Bida GT and Phelps ME (1990b) Acid catalysed thermal decomposition of l-aryl-3,3-diakyl-triazenes in the presence of nucleophiles. J. Org. Chem., 55, 4560-4565. Schlyer DJ, Bastos M and Wolf AP (1987) A rapid quantitative separation of fluorine-18 fluoride from oxygen-18 water. J. Nucl. Med. 28, 764–768. Schlyer DJ, Bastos M, Alexoff D and Wolf AP (1990) Separation of [18F]fluoride from [18O]water using anion exchange resin. Appl. Radiat, Isot., 41, 531–534. Schoeps K-O, Stone-Elander S and Halldin C (1989) On-line synthesis of [11C]nitroalkanes. Appl. Radiat, hot. 40, 261-262. Schoeps K-O, Langstrom B, Stone-Elander S and Halldin C (1991) Synthesis of [l-11C]-D-glucose and [111 C]-D-mannose from on-line produced [11C]nitromethane. Appl. Radiat. Isot., 42, 877–883. Schoeps K-O and Halldin C (1992) Synthesis of racemic [a-11C]amphetamine and [a-11C]phenethylamine from [11C]nitroalkanes.y. Label. Cmpd. Radiopharm. 31, 892-901. Schoeps K-O, Halldin C, Nagren K, Swahn C-G, Karlsson P, Hall H and Farde L (1993) Preparation of [111 C]dopamine, [l- 11 C]p-tyramine and [l-"C]m-tyramine. Nucl. Med. Biol. 20, 669–678. Schrobilgen GJ, Firnau G, Chirakal R and Garnett ES (1981) Synthesis of [18F]XeF2, a novel agent for the preparation of 18F radiopharmaceuticals. J. Chem. Soc. Chem. Comm., 198–199. Shields AF, Lim K, Grierson J, Link J and Krohn KA (1990) Utilization of labeled thymidine in DNA synthesis: studies using PET. J. Nucl. Med., 31, 337–342. Shields AF (1993) Measurement of tumor proliferation using [ 11 C]thymidine and PET. In Clinical PET in Oncology, Proc. of the 2nd Int'l Symp. On PET in Oncology, Sendia, Japan, 41-45. Shiue C -Y, Salvadori PA and Wolf AP(1982) A new synthesis of 2-deoxy-2-[18F]fluoro-D-glucose from 18Flabeled acetyl hypofluorite, J. Nucl. Med., 23(10), 899–903. Shiue C -Y, Watanabe M, Wolf AP, Fowler JS and Salvadori P (1984) Application of the nucleophilic substitution reaction to the synthesis of no-carrier-added [18F]fluorobenzene and other l8F-labeled aryl fluorides. J. Label Cmpds. Radiopharm., 21(6), 533-547. Shiue C -Y, Fowler JS, Wolf AP, Watanabe M and Arnett CD (1985) Synthesis and specific activity determinations of NCA 18F-labeled butyrophe butyrophenone neuroleptics: benperidol, haloperidol, spiroperidol and pipamperone. J. Nucl. Med., 26, 181–186. Shiue C -Y, Bai L -Q, Teng R and Wolf AP (1986a) Application of the nucleophilic substitution reactions to the synthesis of NCA 18F-labeled radioligands. J. Label. Cmpds. Radiopharm., 23, 1038–1039. Shiue C -Y, Fowler JS, Wolf AP, McPherson DW, Arnett CD and Zecca L (1986b) No-carrier-added fluorine-18 labeled N-methylspiroperidol: synthesis and biodistribution in mice. J. Nucl. Med. 27, 226-234.

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8. AUTOMATION FOR THE SYNTHESIS AND APPLICATION OF PET RADIOPHARMACEUTICALS DAVID L. ALEXOFF Department of Chemistry, Brookhaven National Laboratory, Upton, New York 119735000, NY, USA

INTRODUCTION The development of automated systems supporting the production and application of PET radiopharmaceuticals has been an important focus of researchers since the first successes of using carbon-11 (Comar et al., 1979) and fluorine-18 (Reivich et al., 1979) labeled compounds to visualize functional activity of the human brain. These initial successes of imaging the human brain soon led to applications in the human heart (Schelbert et al., 1980), and quickly radiochemists began to see the importance of automation to support PET studies in humans (Lambrecht, 1982; Langstrom et al., 1983). Driven by the necessity of controlling processes emanating high fluxes of 511 KeV photons, and by the tedium of repetitive syntheses for carrying out these human PET investigations, academic and government scientists have designed, developed and tested many useful and novel automated systems in the past twenty years. These systems, originally designed primarily by radiochemists, not only carry out effectively the tasks they were designed for, but also demonstrate significant engineering innovation in the field of laboratory automation. These laboratory automation systems draw heavily on the chemical engineering concepts of unit operations and have evolved from isolated manually operated electro-mechanical devices to large-scale integrated systems utilizing the latest in personal computer (PC) and laboratory robot technologies. The success of these initial engineering efforts carried out by radiochemists is an important reason for the recent growth of clinical PET procedures due to the increased availability of cost-effective PET radiochemicals that commercially available systems now provide. This chapter will first briefly describe the evolution of these automated systems for PET, followed by a discussion of specific engineering design considerations. The scope of this chapter will focus on the design of automated systems for the rapid synthesis and application of PET radiotracers labeled with 15O, 13N, 11C, and 18 F, Systems designed to use other important positron emitting nuclides will be described only in the context of the evolution of engineering design in PET radiopharmaceutical automation. Finally, this presentation will highlight current automated systems addressing automation of both the synthesis of radiotracers for PET, and the assay of radioactivity in plasma for input function determination needed for quantitative PET imaging. Methodological details of specific automated systems including schematic diagrams can be found in an excellent compilation of automated production methods by Crouzel et al., (1993). Automated systems for accelerator, particle beam, and target control will not be discussed. The reader is referred to a recent review Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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article describing integrated, automated accelerator and target systems for clinical PET radiotracer production (Satyamurthy, 1999). AUTOMATION OF RADIOTRACER SYNTHESIS Because of the unique operational and safety requirements of PET radiotracer synthesis, the motivation for the development of automated systems is clear and compelling. These unique constraints include short synthesis (often limited to 2 or 3 half-lives) times and control from behind bulky shielding structures that make both access to, and visibility of, radiochemical processes and equipment difficult. Often curie levels of positron emitting nuclides are required for synthesis of PET radiopharmaceuticals, making this potentially dangerous for a radiochemist or laboratory specialist. The use of short half-lived radionuclides also necessitates that many PET radiotracers (particularly those labeled with 11C, 13N, and 15O) be synthesized repetitively during the day, each dose being produced separately just before administration. Radiotracer synthesis must be reliable and efficient to keep the costs of PET procedures down. Furthermore, radiotracer synthesis procedures for human use must produce pharmaceutical quality products and be well documented and controlled to help satisfy requirements of federal and local regulations on human research. Automation can help PET research institutions overcome all of these potential limitations. A look at the history of the development of successful automated PET radiotracer synthesis machines reveals a richness in engineering solutions to these problems that still exists today. A HISTORICAL PERSPECTIVE: CHEMISTRY FIRST Automated synthesis systems require no direct human participation to perform the various physical and chemical operations that comprise a synthesis. Scientists outside of PET radiopharmaceutical research were the pioneers of automated synthesis, with the most well known example being the work in solid phase peptide synthesis (SPPS) by Merrifield and co-workers (Merrifield et a/., 1966). It was Merrifield's innovations in peptide chemistry that laid the foundation for the development of fully automated commercially available (e.g., Applied Biosystems, Foster City, CA) synthesizers of today. More recently, in 1981, Caruthers and others developed novel solid phase supported DNA chemistry (Beaucage & Caruthers, 1981; Matteucci & Caruthers, 1981) that led to the development of modem DNA synthesizers that were used almost exclusively in mapping the human genome (Caruthers, 1985). Unfortunately, these highly successful automated bench top synthesis systems were designed for a rather narrow range of chemistries and therefore did not lend themselves to adaptation by PET radiochemists for radiosynthesis automation. In general, PET radiosynthesis draws from a broader chemistry knowledge base rooted in synthetic organic chemistry (Fowler & Wolf, 1982; Fowler & Wolf, 1997). However, these examples do serve to make an important point: that the success in synthesis automation requires first and foremost innovative chemistry. Parallel to these important developments in the 70's and 80's in automated oligiopeptide and nucleic acid chemistry was the exploration in automation by traditional synthetic organic chemistry labs. Motivated by the desire to optimize organic synthesis yields efficiently, researchers outside of the field of PET developed the first automated systems for controlling more general-purpose laboratory-scale organic reactions. The control strategy employed by these systems progressed from hard-wired logic control (Legrand & Foucard.

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1978), to microcomputer-based automation (Winicov et al, 1978), to laboratory robot controlled organic synthesis (Frisbee et al, 1984). Development of these systems was motivated primarily by the need to optimize synthetic yields in a synthesis containing several important controllable parameters. These automated organic synthesis systems derive optimum synthesis conditions automatically by applying an optimization algorithm to results obtained from computer and robot controlled experiments (Winicov et al., 1978; Frisbee et al., 1984). Still, progress in automating optimization of organic synthesis reactions had minimal impact on the development of automated machines for PET radiotracer synthesis. The automated synthesis optimization systems often proceeded using standard laboratory equipment using reaction volumes of 50 mL – 5 L. Radiotracer synthesis, on the other hand, is most often carried out in volume range of 5 pL to 5 mL, and has special time constraints and shielding requirements defined by the short-lived isotopes that PET exploits. Hence, as the need to develop automated systems became urgent by the beginning of the 1980's, PET radiochemists were faced with limited or inappropriate functionality provided by automated peptide and DNA synthesis or robot controlled benchtop organic synthesis systems. The design and development of automated radiotracer synthesis systems by PET radiochemists followed a similar evolution to the systems described above, starting as hard-wired, remotely controlled apparatus. Prompted by the success of using the radiopharmaceutical 2-deoxy-2–[18F]fluoro-D-glucose (FDG) to measure localized cerebral (Reivich et al., 1979) glucose metabolism in a living human subject, PET researchers quickly developed synthesis systems that could produce multidose batches of FDG safely, efficiently, and repeatedly (Barrio et al., 1981; Fowler et al, 1981). These systems were manually controlled by manipulator arms and electric switches connected to equipment such as solenoid valves, vacuum pumps, regulated pressure sources, motorized lab jacks, rotary evaporators, and temperature controllers. A skilled operator could manipulate glass vessels, switches, reagents, and solvents from behind the protection of thick lead shielding. Remotely controlled synthesis systems for several carbon-11 compounds were also being developed around the same time (Berger et al., 1979; Padgett et al, 1982; Welch et al, 1982; Welch et al, 1983). Although sometimes referred to as "automated" systems (Berger et al, 1979), these carbon-11 synthesis systems used devices controlled remotely by a human operator in a fashion similar to that described above for the synthesis of FDG (Fowler et al, 1981). The earliest fully automated systems appeared also by the early 1980's. A sampling of these pioneering systems includes hard-wired automatic syntheses of 11C-glucose (Ishiwata et al, 1982) and 13NH3 (Ido & Iwata, 1981), a microprocessor based synthesis of 13NH3 and L-[13N]-glutamate (Suzuki et al, 1982), an automatic production system for the synthesis of 75Br-labelled radiopharmaceuticals (Blessing et al, 1982) based on the Kontron industrial microcomputer (Kontron Embedded Computers AG, Munchen, Germany), and a microcomputer controlled synthesis of the production of FDG (Iwata et al, 1982; Iwata et al, 1984). A closer look at these early systems reveals most of the important underlying characteristics of modern automated systems and how they are designed today. In fact, the complexity and sophistication of radiochemical hardware used in automated PET radiotracer synthesis has not changed significantly since

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these early designs. The greatest progress has come from defining the radiochemical processes themselves and creating the strategies for implementing automatic control. In the automatic synthesis of 11C-glucose (Ishiwata et at., 1982), the radiochemical hardware used for the synthesis of 11C-glucose (19 teflon solenoid valves, 2 reaction vessels, 2 heaters, 4 reagent resevoirs, a vacuum pump, a peristolic pump, 2 Sep-paks™, a purification column and 4 photo-level sensors) reflects accurately the complexity and functionality of hardware used in modem automatic synthesis machines. For example, the FDG machine sold today by Nuclear Interface (Muenster, Germany) has 23 solenoid valves, 1 reaction vessel, 1 heater, 2 Sep-Paks™, 7 reagent reservoirs, a vacuum pump, and two radioactivity sensors. In fact, most automatic radiotracer machines today are configured with two dozen or so valves, 1 or 2 reaction vessels, a heater, a half dozen or so reagent reservoirs, a vacuum pump, and several (or none) sensors for measuring localized radiation fields, vessel pressures, liquid levels, and temperatures.

Methods for automatic control of the physical parameters (pressure differences, temperature, and object displacement) needed to invoke a sequence of steps leading to the synthesis and purification of a particular radiotracer using this generic set of miniature chemistry hardware has evolved greatly in the past two decades. For example, at the same time that Ishiwata and colleagues (Ishiwata et al., 1982) were using hardwired timers, limit switches, and photo sensors to control a 19 step production of llC-glucose, other PET investigators were starting to take advantage of progress in semiconductor technologies leading to the creation of software programmable microprocessors. Suzuki and co-workers (Suzuki et al., 1982) described the automation of the production of 13NH3 and L-(l3N)-glutamate using two general purpose microprocessors (6 kilobytes RAM, 32 kilobytes ROM, 7 digital outputs, 2 analog inputs each). The system described controlled the reaction of 13NH3 with the immobilized enzyme glutamate dehydrogenase (Suzuki et al., 1982) after the reduction of labeled nitrogen oxides with Davarda's alloy and sodium hydroxide. Both software timers and signals from radiation and conductivity sensors were used to control the multistep synthesis. In addition, provisions were made for running the device automatically 4 times without replacing reagents or vessels. Other early microprocessor based systems described how multiple processors could be connected in a distributed control fashion so that more flexible automated systems could be created (Alexoff et al., 1986; Ferdeghini et al., 1987; Russell et al., 1987). These systems, built using 8-bit microprocessors (6511 Rockwell International, Z80 STD Bus Mostek), were designed with the intention of facilitating the automation of multiple radiotracers from a single system. Interfacing and data acquisition responsibilities were separated from broader context control problems like sequencing of steps and display of information. This modular design was intended to make automation of new radiotracer synthesis easier to implement. Microcomputers offer simplified automatic synthesis programming compared to microprocessors (programmed in assembly language) by providing integrated disk, operating system and high-level software language capabilities. One of the earliest applications of the microcomputer to radiotracer synthesis automation was described by Blessing and co-workers (Blessing et al., 1982). This system used the BASIC

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language to supplement assembly language routines (reserved for time-critical operations) to control the isolation of 75Br from a solid target, radiosynthesis, HPLC purification, and solvent evaporation using a rotary evaporator. A unique characteristic of this system was the design of a carrousel type design where the reaction vessel, reagent ports, and refluxing hardware were movable so that plumbing connections could be made in a way that minimized dead volume, valves, and interconnected plumbing. The description of the first automated synthesis of FDG by Iwata et al. represented the first milestone of automation efforts for PET radiopharmaceuticals (Iwata et al., 1984). Iwata's FDG machine was similar in complexity to the automated devices described above and was built from 37 solenoid valves and 18 sensors. This radiochemical hardware was interfaced directly to a microcomputer that was programmed in HP-BASIC to execute sequentially 32 steps comprising the synthesis of FDG following the radiosynthesis described by Fowler et al. (Fowler et al., 1981). This automated system controlled many organic synthesis operations ubiquitous to PET radiopharmaceutical production at the time. These operations included flash column chromatography, solvent evaporation, and radioactivity recovery from purification columns. Iwata's system was the culmination of early automation efforts. Using more than 18 sensors, it was highly instrumented and employed significant feedback control during operation. The system incorporated several types of transducers capable of detecting or measuring liquid levels, gas pressures and flow rates, vessel temperatures, and localized radiation fields. The automated synthesis of 18F-fluoroestradiol by Brodack et al. using a Zymate Laboratory Automation System (Zymark Inc., Hopkington, MA) represented a new approach to PET radiopharmaceutical automation (Brodack et al., 1986). Laboratory robots are interfaced with a microcomputer and can be programmed in high-level languages. Mimicking many human operations and using standard laboratory equipment, these robots could be quickly configured and programmed to carry out a radiopharmaceutical synthesis. In contrast to the highly instrumented "fixed plumbed" machines described previously, early robot-controlled radiosyntheses often lacked the use of feedback sensing to control or monitor specific radiochemical operations like solvent evaporation. The focus of commercial laboratory robot manufactures at this time was in providing robust feedback control strategies for controlling gripping, interchanging hands, and other physical manipulations (Nelson & Lightbody, 1991). By 1990 automated systems were common in many research PET facilities. This was in large part due to the success a novel synthesis of FDG reported by Hamacher et al. (Hamacher et al., 1986). This stereospecific, high yield, one-pot synthesis based on the nucleophilic reagent K+[2.2.2]18F- lead to a proliferation of custom built automated FDG systems at research PET centers (Alexoff et al., 1989; Padgett et al., 1989; Hamacher et al., 1990; Mader et al., 1992) and became the synthetic pathway of most modern commercial FDG machines. The synthesis of FDG by nucleophilic substitution using Kryptofix 2.2.2 not only provided a simple, efficient, stereospecific route, but it also allowed the utilization of new high yield cyclotron targets for the production of 18F- from H218O (Kilbourn et al., 1984; Wieland et al., 1986). During this time PET radiochemists were using a plethora of automatic control strategies. Goodman et al. report the automatic synthesis of 15O-butanol, 15O-water (Goodman et al., 1991a), and 1-11C-

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aminocyclobutane carboxylic acid (Goodman et al. 1991b) using an 8085 microprocessor. Researchers at KFA Julich, Germany also describe an automated oxygen-15 labeled butanol system based on a Programmable Logic Controller (PLC) (described in Crouzel et al., 1993). These systems were designed for up to 8 repetitive syntheses using the reaction of n-butylborane with 15O-O2 on an alumina Sep-Pak™ as first described by Kabalka et al. (Kabalka et al., 1985). In both automated butanol systems, radiopharmaceutical quality formulations for clinical studies were obtainable with simple in-line solid phase extraction (Berridge et al., 1986), further simplifying automation of this efficient, fast reaction. Still other PET radiochemists recognized that the functionality of PLCs matched well the sequential nature of even more complex syntheses of PET radiotracers. A Toshiba EX40 industrial PLC was used by Clark & Dowsett to control the synthesis of a variety of carbon-11 labeled compounds from 11CH3I, including [Omethyl-11C]raclopride, [N-methyl-11C]SCH 23390, and S-[N-methyl-11C]nomifensine (Clark & Dowsett, 1992). At the same time the researchers were using microprocessors and industrial PLCs to automate their processes, Ruth and colleagues describe the use of a personal computer connected to an intelligent data acquisition system (Optomux™, Opto22, Temecula, CA) to synthesize L-6[18F]fluorodopa (Ruth et al., 1991a). A similar control strategy was used by Hamacher et al. in the computer-controlled synthesis of FDG (Hamacher et al., 1990). Both of these systems were programmed in high level languages common to personal computers. Personal computers provide more sophisticated user interfaces and programming capabilities than PLCs or microprocessors that can expedite software development. Finally, a variety of commercial laboratory robot systems were used to synthesize both 11C and 18F labeled compounds by several different groups (Brihaye et al., 1994; Brodack et al., 1988; Brodack et al., 1991). The latest milestone in the development of automated PET radiotracer synthesis machines was reached in the mid 1990's with the report of a high yield, high specific activity gas phase synthesis of 11CH3I (Link et al., 1997; Larsen et al., 1997). Gas phase synthesis of 11CH3I had several advantages over the popular wet chemistry method (Langstrom & Lundqvist, 1976) including rapid turnaround for multiple syntheses and simplified operation for automation. This method was quickly commercialized and evaluated for routine use in PET research environment (Fallis et al., 1997). The commercial system (GE Medical Systems, Milwaukee, MN) was constructed using an industrial PLC with open loop timed control of synthesis steps. Designs of modern automation systems for PET still reflect this richness in automatic control strategy. This diversity is no doubt in part a reflection of the breadth of chemical pathways the PET radiopharmaceutical production relies on, as well as an indication of the individual vitality of each group in the international PET radiochemistry community. UNIT OPERATIONS DESIGN Although the evolution of automated chemistry systems for PET radiopharmaceutical has resulted in a proliferation of designs and control strategies, all of these systems were created using the modular design concept of laboratory unit operations (Padgett et al., 1982: Severns & Hawk, 1984). PET

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radiopharmaceuticais can not only be made using a set of generic hardware of solenoid valves and vessels as just described, but more important, each radiosynthesis can be broken down into a set of common laboratory operations. In radiotracer synthesis these general-purpose operations include manipulations common to the organic chemist like transferring reagents, evaporating solvents, regulating vessel temperature, and solid phase extraction (SPE). Unit operations design was first successfully applied to the design of remotely controlled syntheses of several 11C (Berger et al., 1979; Welch et al., 1982; Padgett, et al., 1982) and 18F labeled compounds including FDG (Barrio et al., 1981; Fowler et al., 1981), This modular approach to remote apparatus construction has a parallel application to the design of automated equipment and computer software (Alexoff et al., 1986; Feliu 1991; Russell et al, 1987). The utility of a unit operations approach is perhaps best demonstrated by laboratory robot systems (Severns & Hawk, 1984). In these robot systems, general purpose workstations like solid phase extraction or reaction vessel heating surround a central manipulator arm which can be programmed to execute unique sequences of steps to create a specific automated process like a radiotracer synthesis. In this instance, unit operations are distinct not only functionally, but also by physically separate, disconnected pieces of hardware. In nonrobotic automated systems, unit operation boundaries are defined more algorithmically, although concomitant hardware exists and is plumbed together (Padgett et al., 1982). In these fixed-plumbed automated systems, modular designs are used for intelligent interface hardware that connects laboratory and synthesis equipment to a controlling computer or microprocessor as well as software design of highly structured synthesis software. In fact, many modern laboratory automation machines use object oriented programming languages like Visual Basic (Cadavid et al., 1997) that facilitate the creation of highly structured and modular automation tools (Echols & Russon, 1997; Feiglin & Russell, 1997). In sum, although sometimes confused with the concept of fixed-plumbing automation, unit operations is a useful engineering concept that has been applied successfully to both robot systems (Brodack et al., 1988; Brihaye et al., 1994; Brihaye et al., 1996; Krasikova, 1998) and fixed plumbed "black-box" automation (Satymurthy et al., 1999). Structured design techniques facilitate the development of automated systems from manual methods by first providing plumbing building blocks for remotely-controlled systems (Clark & Dowsett, 1992; Crouzel et al., 1993) and then providing a framework for both process control system and software design (Alexoff et al., 1986; Russell et al., 1987). The decision to use modular hardware and software design can mitigate the cost and time needed to develop new automated systems by providing generic solutions to focussed control problems (such as the evaporation of solvent from a reaction vessel or the isolation of a component by SPE) found in radiotracer syntheses. Given a complete enough set of generic automation building blocks, the automation of any radiotracer sythesis could be carried out by radiochemists with a minimum of automation expertise (Alexoff, 1991; Felieu, 1991). This flexibility of both modern robot and fixed-plumbed automated systems in PET contrasts the one-of-a-kind nature of early hardwired automated systems built by PET radiochemists.

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ROLE OF FEEDBACK CONTROL While most PET radiochemists involved in automating their processes will agree on the virtues of the concept of unit operations and modular design, the extent of the use of feedback control in both custom and commercial automated systems has varied greatly. The utility of including sensors for feeding back information during synthesis has been debated (Link & Clark, 1994). Most of the arguments reflect concerns about reliability. For example, the well-established engineering design principle of "keep-it-simple" dictates that before the added complexity of incorporating sensors can be justified they must provide functionality and information that increases reliability and overall performance. In fact, one of the first successful commercial FDG machines was first designed with feedback control, only to be marketed without sensors required for closed loop control of unit operations (Satyamurthy et al., 1999). The following quote from a one of the developers of the prototype machine summarizes well the debate over incorporating sensors in automated PET radiopharmaceutical equipment: "The initial module incorporated self-diagnosis and feedback from sensors such as vapor pressure monitors, liquid level sensors, etc. However, the system worked quite well with a simple series of on/off commands and time waits. Thus, to maintain simplicity and reliability, the time of various tasks that took place during the synthesis was determined and a margin for variation incorporated in the final program." Although many of the automated systems already described sequence the steps required to carry out radiotracer synthesis in the same open loop timed control strategy, it can be argued that appropriate feedback control strategies can increase reliability by automatically compensating for dynamic process variables. For example, variable volumes of solvent to be evaporated could change drying times considerably. Without any feedback, an evaporation step time would have to be set for the longest evaporation time (largest solvent volume). Alternatively, volume information could be input to the system and a previously calibrated lookup table mapping drying times to solvent volumes could be used to determine an appropriate evaporation time. This could be extended to account for changes in solvent composition. Even so, this strategy would require feedback from either an operator or an appropriate liquid sensing system. Fortunately, most processes automated for PET radiotracer production have well-defined parameters that lend themselves to open loop control strategies. It is clear from this example that with the use of appropriate sensors and feedback control algorithms, more robust, general-purpose machines can be built. Furthermore, information gathered from sensors can be important for either pre-run diagnostics (Alexoff et al., 1986; Iwata et al., 1990) or computer assisted problem solving (Alexoff, 1991). Advanced features like these may be critical to future development of commercial 11C labeled radiotracer machines, where it is even more important to minimize synthesis times, provide reliable control, and simplify operation and maintenance. In fact, the trend in modem automated systems is to include such feedback strategies (Jackson, 2000; Zigler, 2000). REAL-TIME CONTROL OF UNIT OPERATIONS Feedback control strategies for PET radiotracer synthesis control can be classified as either continuous (regulatory) or discrete (step control). In continuous control, sampled data from process sensors is input to

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an algorithm that modulates an output device to achieve a desired value (setpoint) of the measured process variable. For example, reaction vessel temperature regulation is often achieved by using a Proportional Integral Derivative (PID) control algorithm that is either part of a separate commercial controller (Mader et al., 1992) or synthesis control software (Alexoff et al., 1989). Besides being used for feedback control of temperature regulation, regulatory type control in PET radiotracer manufacturing systems has been limited to a small number of applications, mostly gas flow systems. For example, Le Bars et al. described a PLC system that automatically adjusts a dilution gas flow rate to regulate a final radioactivity concentration flow to the patient. By including a feedback circuit that regulated gas radioactivity concentration, these designers minimized changes in radioactivity delivered to the patient due to disturbances in particle beam irradiation conditions, including momentary disruption of beam (Le Bars et al., 1991). Most unit operations for PET tracer synthesis do not require regulatory type feedback control action, but represent instead discrete or threshold type control problems. More recently, step control using a variable threshold or fuzzy logic approach has been proposed (Hichwa, 2000). Fuzzy logic strategies attempt to mimic human decisions by incorporating production trends or other information available to human operators (see also Alexoff, 1991). Most feedback control discussions in PET tracer synthesis have focussed on the utility of using sensors in this way to determine the status of discrete steps comprising a synthesis, whether it be a fixed threshold control or more sophisticated fuzzy logic approach. Solvent evaporations and liquid transfers are the two most common unit operations used in automated PET systems that have been subject to feedback control using sensors. These operations often represent more than 90% of control responsibilities comprising the execution of a typical radiotracer synthesis. At Brookhaven Lab, for example, an automated synthesis of FDG proceeds in 36 discrete steps, 5 of which are solvent evaporations, 22 of which are liquid transfers of some sort (extractions, vessel washes, transfers, etc.). Solvent evaporations and liquid transfers can each be subdivided into two subtypes, each posing a slightly different control problem. Solvent evaporations, for example, may be used for drying or concentration. Liquid transfers, particularly for solvent delivery, are used either to move fixed volumes of liquid from one place to another or to dispense a programmable volume. While control of both drying and batch transfer require information about when a discrete volume of liquid is either evaporated or transferred respectively, concentration and dispensing control require feedback about the remaining volume in a reaction vessel of solvent reservoir. Most sensor applications have been developed for drying or batch transfer control, although feedback control of dispensing solvent using a mass flow controller during synthesis has also been described. (Iwata et al., 1990). Solvent evaporations Conductivity (Link et al., 1994), temperature (Link et al., 1994; Zeisler et al., 1994) and solvent vapor pressure signals (Ducret et al., 1994) have all been used as feedback signals to drying algorithms in automated synthesis equipment. Gas vapor pressure signals are obtained directly through pellistor type gas sensors (Ducret et al., 1994) or indirectly using diaphragm type pressure transducers (Alexoff et al., 1989). The most common feedback practice, however, is to use encapsulated thermistor or thermocouple inserted inside the reaction vessel. Robust signals for input to drying algorithms for both aqueous (Zeisler et al.,

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1994) and organic (Link et al., 1994) solvents are obtained by the effect of evaporative cooling on temperature sensors in liquids. The most common control algorithms include a simple comparison of current process variable values with empirically determined "dry" endpoint values (Link et al., 1994). To help eliminate premature endpoint triggering due to electronic noise, signal averaging of sampled data in digital systems is often used before endpoint testing in software (Link et al., 1994). In addition, simultaneous smoothing and differentiation using simple but robust digital filters (Savitzky & Golay, 1964) can provide drying control information for algorithms that are less dependent on empirical endpoints that could be susceptible to change. For example, thresholds based on rates of change are less dependent on absolute drying conditions and can be used in conjunction with signal magnitudes to create robust endpoint determination algorithms for solvent evaporation (Alexoff et al., 1986). Fluid transfers Since the earliest prototype automated systems, liquid sensing has been employed. Reservoir liquid levels can be monitored by optical detectors mounted exterior to the reservoir that give a digital signal indicating the presence of a liquid at some predefined level. Liquid presence sensors for tubes can be used to determine whether a tube is filled with a liquid or not. Most often these detectors rely on changes in reflected or transmitted light emanating from a solid state or incandescent energy source due to a change in refractive index inside the vessel filled with liquid compared to air or inert gas (Zeisler et al., 1994; Alexoff et al., 1994). A different type of liquid presence sensor has been designed to take advantage of changes in dielectric constant of fluids (e.g., water vs air in a tube) (McKinney et al., 1995). This design greatly improves the radiation hardness of the liquid detectors used in automated radiopharmaceutical production equipment (McKinney et al., 1995). Note that these sensing strategies are limited to discrete type control problems associated with determining when the transfer of a fixed volume of liquid has been completed. Other methods of determining the completion of a liquid transfer also have the potential of continuous control for dispensing applications. A method reported by Iwata et al. based on thermal mass flow controller can be used for both liquid transfer and liquid dispensing applications. In this feedback control strategy, measurements of instantaneous gas flow rate can be used to assess the completion of a liquid transfer while a real time integration of transfer gas flow rate can be used to dispense calibrated volumes of liquid (Iwata et al., 1990). Another advantage of this system is that, depending on the plumbing of a specific automated system, a single sensor can be used as feedback to control transfer and/or dispensing tasks from multiple reagent vessels or reservoirs. In a similar fashion, changes in pressure measured by a pressure sensor have also been shown to give robust signals indicating the completion of liquid transfers associated with solid phase extraction (Alexoff et al., 1989). In this example, the changes in resistance due to the presence then absence of liquid between the vent and the vacuum source give rise to decreased pressure differentials across valves and tubing that signal clearly the completion of batch liquid transfers. Radioactivity sensors can also be used to provide feedback for liquid transfer control (for detector examples, see Crouzel et al., 1993). Radioactivity measurements for fluid transfer control can be especially useful when controlling the release and transfer of small volumes of gas, from, for example, irradiated cyclotron targets. These small volumes are often introduced with a carrier gas stream under a constant flow rate into a

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reaction vessel or trap. In this case, radioactivity may be the only measure, even in manual or remotely controlled syntheses, that allows an operator or algorithm to determine the end of the gas transfer. Ruth and co-workers have presented an example where 11CO2 is released from a liquid nitrogen trap into a reaction vessel (Ruth et al., 1989). In this example, transfer signal endpoints derived from integrated radioactivity signals can be used for transfer control (Ruth et al., 1991b). SENSOR DATA FOR DIAGNOSTICS AND DOCUMENTATION The trend in automated radiopharmaceutical synthesis is proceeding steadily to include more feedback control. For example, many second-generation commercial machines have some kind of feedback (e.g., Jackson, 2000; Zigler, 2000). One of the motivations for this is not only more robust and efficient real time control as just discussed, but as importantly, information from process sensors aids in pre-ran diagnostics, post-run troubleshooting, trend analysis, and process documentation. Pre-run diagnostics built into automated PET radiotracer equipment include signal noise measurements, leak rates of vessels, heater performance, and automated PID tuning (Alexoff et al., 1986). Iwata described the use of a mass flow controller to assess the presence of liquid in a vessel by measuring the head-space in the vessel from gas flow rate measurements (Iwata et al., 1990). Pre-run diagnostics still rely on careful operator inspection of liquid levels, tubing connections, and vessel connections. This visual inspection is often aided by automated or computer-assisted leak checking. Sensor information is also important to follow manufacturing trends in a radiopharmaceutical production line. It is often possible for an experienced radiochemist to respond to subtle changes in precursor yield, age of reagents, or integrity of radiochemical hardware to maintain overall synthesis yields. Although the increased availability of PET synthesizers using totally disposable components (Mosdzianowski & Morelle, 2000) can help to minimize some of these problems, data from sensors can be used to schedule important maintenance of radiochemical equipment. For example, Ferrieri et al. have recently demonstrated that the strategic placement of a single radiation detector external to the GEMS methyl iodide box can give useful information about the integrity of a major reagent supply (I2 tube) used to make 11C-rnethyl iodide (Ferrieri et al., 2000). Robust changes in radioactivity signal frequency, integrated activity, and overall curve shape (rates of changes and inflection points) are observed as the I2 tube ages. These changes in radioactivity signal characteristics can be used to schedule preventative maintenance tasks, thus avoiding unexpected radiolabeling failures due to low 11C-methyl iodide yields. Other PET researchers have reported the use of trend data from an in-line conductivity sensor upstream of automated synthesis equipment to schedule target and delivery line maintenance (McKinney, 2000). This scheduled maintenance of important radiochemical systems feeding automated synthesizers avoids unexpected decreases in radiotracer yield due to changes in precursor purity, delivery time, and delivery line losses that are a function of cumulative target and delivery line use. (McKinney, 2000). Incorporating automated or computer-assisted troubleshooting capabilities into PET radiotracer synthesis machines has been proposed as a solution to the problem of the disparity of knowledge and experience of

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OEM designers compared to end users (Alexoff, 1991). Automated troubleshooting may also be useful for institutions using in-house machine designs by empowering less experienced operators with the knowledge of equipment designers and veteran radiochemists. It is clear that incorporating more sophisticated software strategies such as artificial intelligence based troubling-shooting or fuzzy logic unit operations control requires the increased use of sensors. Although advances in radiotracer chemistry will continue to provide simple and robust systems that minimize the need for increased intelligence of PET radiotracer synthesis machines, the development of sophisticated synthesizers with optimal control and autodiagnostic capabilities could facilitate the supply of cost-effective new radiotracers for clinical use. MODERN AUTOMATIC SYNTHESIZERS Design concepts for modern automatic synthesizers for PET have been discussed and various approaches to automation have been compared and contrasted (Crouzel et al., 1993; Link et al., 1992; Satyamurthy et al., 1999). Some of the important design criteria to consider when building an automated synthesis system include multi-run capability, requirements for sterile disposable components, self-cleaning capability, autodiagnostic functions, and process documentation. These design criteria effect both the choice of specific radiochemical process control hardware (e.g., valve type or tubing material) and overall control system design (e.g., robot, PC, PLC). Most modern machines share a highly structured, modular, unit operations based design of radiochemical processes, valve and tubing hardware, and intelligent (computer/microcomputer based) data acquisition and control hardware and software systems. Today's machines utilize highly modular, distributed intelligent industrial process control and data acquisition hardware such as OPTO-22™ (Opto22, Temecula, CA) and Fieldpoint™ (National Instruments Corporation, Austin, TX). These systems are modular and expandable, providing appropriate input/output (I/O) densities of common industrial I/O hardware (e.g. medium power DC output, PID control, analog to digital (A/D) conversion, analog filtering) for machine designers. These intelligent interface systems are often optoisolated for high noise immunity, allowing user interfaces (PC) to be located large distances from the actual control area (e.g., shielded synthesis hood). Additionally, modern software engineers have available to them a rich palate of graphically based, object oriented user interfaces and software tools such as Lab View™ (National Instruments Corporation, Austin, TX), FactoryFloorTM22TM (Opto22, Temecula, CA), and Visual BasicTM(Microsoft, Seattle, WA). A brief discussion of two modern machines serves to highlight these latest engineering design strategies as well as to illustrate the diversity of engineering solutions to the problem of automated PET radiotracer synthesis that persists today. The reader is also directed to the web sites and product specifications of the major commercial suppliers of automated radiochemical production equipment (Sumitomo, CTI, Concurrent Microsystems, Nuclear Interface, GE, Ebco). AUTOMATED SYNTHESIS OF 6-[18F]FLUORO-L-DOPA Significant engineering innovation is demonstrated in the automated synthesis of 6-[18F]fluoro-L-DOPA reported by de Vries et al. (de Vries et al., 1999). Success of this machine depends first and foremost on the choice of synthetic route. As the authors discuss, the choice of electrophilic fluorodestannylation as a synthetic pathway gave high yields of labeled compound without the complication of labeled isomers that

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require separation. Furthermore, the route chosen did not require separation of a labeled intermediate and therefore allowed the synthesis to proceed in one pot. Finally, HPLC conditions were such that evaporation and reformulation of the purified product was not required. This work demonstrated that simplified chemical processes amendable to automation could be implemented without compromising radiopharmaceutical quality. This successful system was constructed by modifying a commercially available PET radiotracer synthesizer (Nuclear Interface, Muenster, Germany) that was designed for the automated synthesis of FDG. Success of this system is a testimony to the flexibility of most modern radiotracer systems that use structured software designs, modular intelligent interfaces, and unit operations-based radiochemical processing. Utilizing pressure, radioactivity, and UV sensors incorporated in the commercial machine, the authors present robust process signals documenting most steps in the entire process (de Vries et al., 1999). Although it is not clear that these signals are used directly as feedback for step control, they do provide important process documentation and information for troubleshooting. As described in this work, the Nuclear Interface machine also includes automated cleaning and automated diagnostics for flow and leak checking. Finally, a fluid sensor detecting the presence of liquid in tubing leading to the HPLC injector was incorporated so that HPLC injection could be automated. An interesting finding of the authors was the failure of the fluid sensor due to a sensitivity to metal ions used during neutralization of HBr used for hydrolysis. Proper function of the sensor was restored by changing the reagent used for neutralization from 10N NaOH to 25% ammonium hydroxide with phosphate buffer. In this instance, successful application of feedback control required a commitment of the radiochemists and modification of chemical processing to accommodate sensor characteristics. ROBOT SYNTHESIS OF [11C]FLUMAZENIL Krasikova et al. report the use of a commercially available Anatech RB-86 robot (Anatech, Husbyborg, Uppsala, Sweden; for detailed description see Krasikova, 1998) to prepare [11C]flumazenil from [11C]methyl iodide (Krasikova et al., 2000). This laboratory robot system has also been used to automate other PET radiotracer syntheses including FDG and L-[C-11 -methyl]methionine (Krasikova, 1998) and includes a personal computer (PC) and programmable logic controller (PLC). Robot workstations include hardware for solid phase extraction (SPE), solvent evaporation, and reaction vessel capping/dilution. Starting from trapped [11C]CH3I, the synthesis of labeled flumazenil proceeds in just 7 steps and is completed in 18 minutes. A novel feature of this system was the elimination of HPLC purification. Although HPLC injection and purification can be automated reliably (see above), alternative purification strategies can simplify control and shorten overall synthesis times considerably. This is especially important for the synthesis of carbon-11 compounds. In this work, the authors demonstrated that through the careful determination of optimal conditions for both solid supported alkylation of the desmethyl compound Ro 15-5528 using [11C]CH3I, and the separation of [11C]flumazenil from Ro 15-5528, HPLC purification could be eliminated altogether. Krasikova et al report a mass of Ro 15–5528 in the final product formulation of [11C]flumazenil (7.5 mL) to be only 0.1 to 0.8 micrograms using this method (Krasikova et al., 2000).

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It is clear from the success of these two different automatic control designs that consideration of a diversity of machine designs by radiochemists is appropriate when faced with the challenge of automating the synthesis of a PET radiopharmaceutical. This design diversity reflects the unique challenge facing PET radiochemists who draw upon the myriad of strategies and pathways inherent to organic chemistry. In fact, the power of the PET method in research is derived in part from this basis in organic chemistry and the concomitant plethora of biologically important molecules that can be labeled with the positron emitting nuclides 18F, 11C, 13N, and 15O. Automated synthesis designers must be prepared to incorporate this flexibility when building machines in support of PET research. AUTOMATION FOR THE APPLICATION OF PET RADIOPHARMACEUTICALS Development of automated systems for PET research has not been limited to the design of machines to carry out the syntheses of radiopharmaceuticals. Motivated by many of the same problems presented by routine, rapid syntheses of PET radiopharmaceuticals, PET researchers have also developed automated systems to facilitate the application of PET radiotracers in basic and drug research and development. These systems include automated quality control of radiotracers (see Crouzel et al., 1993), computer controlled infusion systems for automated injection of radiopharmaceuticals (Palmer et al., 1995), automated dose dispensing systems (Jackson, 2000; Plascjak et al., 1997), and automated delivery of radiotracers using pneumatic transport systems (Dembowski & Gonzalez-Lepera, 1994). In particular, significant progress has been made in automating plasma analyses required for quantitative PET studies (Alexoff et al., 1995; Andersson & Schneider, 1998; Lindner et al., 1995; Luthra et al., 1992). Accurate assays of unchanged PET radiotracers in plasma (plasma input function) are important for the determination of model parameters that reflect specific biochemical properties of specific molecular targets (e.g., receptor availability or enzyme concentration). Determination of these input functions can be time consuming, labor intensive, as well as hazardous. In certain instances, input functions can be generated noninvasively using reference tissue regions (Logan et al., 1996). In general, however, new tracers are validated and new drug research is carried out with direct measurements of plasma radioactivity and its identity. These measurements are often carried out for multiple blood samples making up a discretely sampled function representing the time-course of radiotracer activity after bolus injection. Automated blood sampling devices (Grahm & Lewellen, 1993) may be used to obtain discrete blood samples for automated analysis. Flow counting systems generating continuous time-activity data have also been used to automate input function measurement, particularly in 15O studies (Hutchins et al., 1986). Automated systems have been described for automating plasma assays for unchanged radiotracer in plasma using laboratory robots (Alexoff et al., 1995; Andersson & Schneider, 1998), and programmable logic controllers (Luthra et al., 1992). The latter system is based on HPLC and automates extraction of radioactivity from plasma followed by analysis by HPLC and therefore may be applied to any suitable HPLC method for any new radiotracer. The system requires only one person to operate (manual injections) and has been used successfully to determine the unchanged fraction of radiotracer in plasma for several compounds including 11C-L-deprenyl, 11C-diprenorphine, 11 C-flumazenil, 11C-raclopride, and 11C-SCH 23390. By contrast, the laboratory robot system described by BNL researchers to automate the same task requires no

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human participation. This system, however, is based on a validated solid phase extraction assay that eliminates HPLC and therefore may not be as universally applied to new radiotracer assays without revalidation. Selective assays for many radiotracers, however, have been developed and implemented using this robotic SPE-only strategy (Alexoff el al., 1996). Although performance of these automated systems is often reliable (a 6 year "uptime" in excess of 95% is reported by Andersson & Schneider, 1998), sample throughputs for the plasma assay robots can be 1/3 to 1/2 throughputs achieved by an experienced human laboratory worker. At BNL, for example, robotic steadystate throughput of 14,3 samples/hour (2 minute counting interval) is 1/3 that of a human worker (1 minute counting interval). As first reported, this throughput rate is highly dependent on the range of whole blood volumes in a study because of an iterative gravimetric feedback algorithm used to obtain cell-free plasma for counting. This algorithm uses a linearized model of a 1.5 mL tapered blood sampling tube (Eppendorf) and an initial estimate of the subjects hematocrit and a maximum whole blood volume to calculate the cell/plasma interface in each tube. Using this technique, the robot's pipetting hand (1.0 mL syringe tip) was able to obtain sufficient cell-free plasma for good counting statistics using as little at 0.2 – 0.4 mL of whole blood (Alexoff et al., 1997). Sample throughput of the system, however, depends strongly on sample volume uniformity. This is usually not a problem when using auto-sampled blood. Using the Anatech RB-86 robot and a direct measure of the cell/plasma interface with an optical sensor, Andersson & Schneider report a throughput of 21 plasma samples/hour (30 second counting interval). This system also incorporates whole blood counting and bar coding of samples, but requires larger whole blood volumes (1–1.5 mL). Direct detection of the cell/plasma interface and the use of several cross-calibrated well counters allows for higher sample throughputs that are independent of sample volume (Andersson & Schneider, 1998). FUTURE DIRECTIONS As illustrated by past successful automated chemistry systems both within and beyond the field of PET radiochemistry, future advances in automated systems will once again reflect mostly the creativity of PET radiochemists and their ability to refine processes and characterize new radiolabeling pathways. Recently, PET radiochemists have continued this tradition by exploiting captive solvent techniques and solid phase reaction schemes to create very simple high yield radiochemical systems that are amendable to automation (Jewett & Kilbourn, 1999; Wilson et al., 2000; Iwata et al., 2000; Iwata et al., 2001). In particular, captive solvent techniques have recently been used by Wilson et al. to make C-11 labeled raclopride, Nmethylspiperone, Ro 15-1788, FLB 457, Rolipram, SCH 23390 and SKF 82957 from [11C]-iodomethane (Wilson et al., 2000). This method extends the pioneering work of Jewett and co-workers (Jewett et al., 1991) by eliminating the need for solid supports and elevated temperatures. This streamlined "loop method" yields efficient trapping of 11CH3I and fast methylation reactions both at room temperature, greatly simplifying radiochemical processing. Recently, researchers in Japan have investigated the use of the "loop method" with [11C]methyl triflate in the radiosynthesis of [11C]raclopride (Iwata et al., 2001). Using this method, an automated synthesis system (starting from [11C]methyl iodide) can be constructed with only 4 valves, I reservoir, a furnace, and an HPLC system (Iwata et al., 2001).

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In addition to impacting future advances in radiolabeling and purification, solid phase techniques can be expected to continue to simplify radiochemical processing that is needed for the formulation of radiopharmaceuticals. Methods based on a C18 Sep-Pak™are being developed to replace the need for rotary evaporators. Lemaire and colleagues report formulations of several C-l1 and F-18 radiopharmaceuticals in 3-6 minutes with recoveries >97% using only solid phase extraction techniques (Lemaire el al., 1999). Taken together, these new strategies for radiolabeling, purification, and formulation of PET radiopharmaceuticals are likely to be utilized extensively in future automated systems, particularly for carbon-11 labeled compounds. PET radiochemists will continue to use the latest in personal computer, industrial control, and laboratory robot technologies to implement these radiochemical processes and others to create reliable, flexible, automated chemistry systems.

SUMMARY It is clear that this current state of reliable, cost-effective commercially available PET radiochemicals is the result of the early engineering groundwork put down by a handful of pioneering radiochemists from around the world. These early pioneers had not only the prescience to see the benefits of automating their processes, but also had the vision to see the benefits of international collaboration. Those of us in the PET field today are greatly in debt to these early innovators whose world-view and breadth of knowledge has put the future of PET on firm ground for this the 21st century.

ACKNOWLEDGEMENTS This work was carried out at Brookhaven National Laboratory under contract DE-AC02-98CH10886 with the US Department of Energy and Office of Biological Environmental Research.

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REFERENCES Alexoff DL, Russell JAG, Shiue C-Y, Wolf AP, Fowler JS and MacGregor RR (1986) Modular automation in PET tracer manufacturing: Application of an autosynthesizer to the production of 2-deoxy-2[l8F]fmoro-D-glucose. Appl. Rad. Isot.,31, 1045–1061. Alexoff DL, Shiue C-Y, Blessing G, Schlyer DJ and Wolf AP (1989) Process signals to control radiotracer syntheses: Experience with the automated production of 2-deoxy-2-[18F]fluoro-D-glucose. J, Label. Comp Radiopharm., 26, 462–464. Alexoff DL (1991) Knowledge-based automated radiopharmaceutical manufacturing for positron emission tomography. In New Trends in Radiopharmaceutical Synthesis, Quality Assurance, and Regulatory Control, Emran AM (ed), Plenum Press, New York, pp. 339-353. Alexoff DL, Hallaba K, Schlyer D and Ferrieri R (1994) A simple liquid detector for radiopharmaceutical processing systems. In Proceedings of the 5th Workshop on Targetry and Target Chemistry, Dahl JR, Ferrieri R, Finn R and Schlyer DJ (eds), Upton, New York, pp. 261-264. Alexoff DL, Shea C, Fowler JS, King P, Gatley SJ, Schlyer DJ and Wolf AP (1995) Plasma input function determination for PET using a commercial laboratory robot. Nucl. Med. Biol., 22, 893–904. Alexoff DL, Shea C, Fowler JS, Gatley SJ and Schyler DJ (1996) Development of robotic plasma radiochemical assays for positron emission tomography. In Proceedings International Symposium on Laboratory Automation and Robotics 1995, Zymark Corporation, Hopkinton, Massachusetts, pp. 628643. Alexoff DL, King P and Gatley SJ (1997) Robotic control of whole blood processing in functional brain imaging research. In Proceedings International Symposium on Laboratory Automation and Robotics 1996, Zymark Corporation, Hopkinton, Massachusetts, pp. 820-826. Andersson JLR and Schneider H (1998) Design, construction and six years' experience of an integrated system for automated handling of discrete blood samples. Euro. J. Nucl. Med., 25, 85–90. Barrio JR, MacDonald NS, Robinson GD, Najafi A, Cook JS and Kuhl DE (1981) Remote, semiautomated production of F-18 labeled 2-deoxy-2-fluoro-D-glucose. J. Nucl. Med., 22, 372–375. Beaucage SL and Carathers MH. (1981) Studies on nucleotide chemistry V. Deoxynucleoside phosphoramidites a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters, 22, 1859. Berger G, Maziere M, Knipper R and Comar D (1979) Automated synthesis of carbon-11 labeled radiopharmaceuticals: imipramine, chlorpromazine, nicotin and methionine. Intl. J. Appl. Rad. Isot., 30, 393-399. Berridge MS, Franceschini MP, Tewson TJ and Gould KL (1986) Preparation of oxygen-15 butanol for positron tomography. J. Nucl. Med., 27, 834–837. Blessing G, Coenen HH, Hennes M and Lipperts H (1982) A computer controlled automatic apparatus for radiochemical separation of 75Br and synthesis of 75Br-labelled radiopharmaceuticals. J. Label. Comp. Radiopharm., 19, 1333-1335.

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Hichwa R (2000) Radiopharmaceutical automation: Sensors and technology vs the human experience. In Proceedings of the 8th Workshop on Targetry and Target Chemistry, McCarthy TJ (ed), St Louis, pp. 59-60. Hutchins GD, Hichwa RD and Koeppe RA (1986) A continuous flow input function detector for H215O blood flow studies in positron emission tomography. IEEE Trans. Nucl. Sci., 33, 545–549. Ido T and Iwata R (1981) Fully automated synthesis of 13NH3. J. Label. Comp. Radiopharm., 18, 244–246. Ishiwata K, Monma M, Iwata R and Ido T (1982) Automated photosynthesis of 11C-glucose. J. Label. Comp. Radiopharm., 19, 1347–1349. Iwata R, Takashashi M, Shinohara M and Ido T (1982) Fully automated synthesis system of [18F]-2-deoxy-2fluoro-D-glucose. J. Label. Comp. Radiopharm., 19, 1350–1351. Iwata R, Ido T, Takahashi T and Monma M (1984) Automated synthesis system for production of 2-deoxy[18F]fluoro-D-glucose with computer control. Appl. Rod. Isot., 35, 445–454. Iwata R, Ido T and Yamazaki S (1990) Intelligent control of liquid transfer. In Proceedings of the 3th Workshop on Targetry and Target Chemistry, Ruth T (ed), Vancouver, Canada, pp. 137–139. Iwata R, Pascali C, Bogni A, Horvath G, Kovacs Z, Yanai K and Ido T (2000) A new, convenient method for the preparation of 4-[18F]fluorobenzyl halides. Appl. Rad. Isot., 52, 87–92. Iwata R, Pascali PC, Bogni A, Miyake Y, Yanai K and Ido T (2001) A simple loop method for the automated preparation of [11C]raclopride from [11C]methyl inflate. Appl. Rad. Isot., 55, 17–22. Jackson M (2000) Developments in FDG synthesis and production. In Proceedings of the 8th Workshop on Targetry and Target Chemistry, McCarthy TJ (ed), St Louis, pp. 73-74. Jewett DM, Mangner TJ and Watkins GL (1991) Captive solvent methods for fast, simple carbon-11 radioalkylations. In New Trends in Radiopharmaceutical Synthesis, Quality Assurance, and Regulatory Control, Emran AM (ed), Plenum Press, New York, pp. 387–391. Jewett DM and Kilbourn MR (1999) High performance extraction disks for the preparation of a PET agent: a fast synthesis of [11C]carfentanil. J. Label. Comp. Radiopharm, 42, S873-S874. Kabalka GW, Lambrecht RM, Sajjad M, Fowler JS, Kunda SA, McCullum GW and MacGregor R (1985) Synthesis of [15O]-labelled butanol via organoborane chemistry. Appl. Rad. Isot., 36, 853–855. Kilbourn MR, Hood JT and Welch MJ (1984) A simple 18O-water target for I8F production. Intl J. Appl. Rad. Isot., 35, 599–602. Krasikova R (1998) Automated synthesis of radiopharmaceuticals for positron emission tomography. Radiochem., 40, 364–372 Krasikova R, Fedorova O, Korsakov M, Nagren K, Maziere B and Halldin C (2000) A fast and convenient method for robotic preparation of [11C]flumazenil avoiding HPLC purification. J. Label. Comp. Radiopharm., 43, 613–621. Lambrecht RM (1982) Production and radiochemical process control for short-lived medical radionuclides. In Applications of Nuclear and Radiochemistry, Lambrecht RM and Morcos N (eds), Pergamon Press, New York, pp. 5–14. Langstrom B, Clark JC, Lindback S and Welch MJ (1983) Automated synthesis of radiopharmaceuticals labeled with short-lived positron-emitters. In Proceedings of the 3rd World Congress on Advances in Nuclear Medicine and Biology 1982, 3, Raynaud C (ed), Pergamon Press, Paris, pp. 2461–2464.

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targets for producing F fluoride with low energy protons. J. Label. Comp. Radiopharm., 23, 1205i 207. Wilson AA, Garcia A, Jin L and Houle S (2000) Radiotracer synthesis from [11C]-iodomethane: a remarkable simple captive solvent method. N. Med. Biol., 27, 529–532. Winicov H, Schainbaum J, Buckley J, Longino G, Hill J and Berkoff CE (1978) Chemical process optimization by computer — a self-directed chemical synthesis system. Analyt. Chim. Acta, 103, 469– 476. Zeisler SK, Ruth TJ, Rektor MP and Gschwandtner GA (1994) Detectors and transducers for target operations and automated P.E.T. chemistry. In Proceedings of the 5lh Workshop on Targetry and Target Chemistry, Dahl JR, Ferrieri R, Finn R and Schlyer DJ (eds), Upton, New York, pp. 249–255 Zigler S (2000) The development of an FDG module designed for the distribution setting. In Proceedings of the 8th Workshop on Targetry and Target Chemistry, McCarthy TJ (ed), St Louis, pp 69.

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9. DESIGN AND SYNTHESIS OF 2-DEOXY-218 18 [ F]FLUORO-D-GLUCOSE ( FDG) JOANNA S. FOWLERA AND TATSUOIDOB A

Chemistry Department, Brookhaven National Laboratory, Upton NY 11973 Tohoku University, Sendai, Japan

B

INTRODUCTION The first synthesis of 2-deoxy-2-[18F]fluoro-D-glucose (18FDG) for human studies took place in 1976 the result of a collaboration between scientists at the National Institutes of Health, the University of Pennsylvania and Brookhaven National Laboratory which had begun three years earlier. 18FDG was developed for the specific purpose of mapping glucose metabolism in the living human brain thereby serving as a tool in the basic human neurosciences (Ido et al., 1978; Reivich et al., 1979). With 18FDG it was possible for the first time to translate the [14C]2-DG autoradiographic method (Sokoloff, 1979) to the clinical arena. Around the same time that 18FDG was developed, preclinical studies suggested the utility of 18FDG for studies of myocardial metabolism (Gallagher et al., 1977) and for tumor metabolism (Som et al., 1980). In the first human studies and many that followed, 18FDG was synthesized at Brookhaven National Laboratory on Long Island and sent by small plane to Philadelphia Airport and then transported to the Hospital of the University of Pennsylvania where the first images of a human volunteer were made (Figure 1).

In spite of the 110 minute half-life of fluorine-18 and the relatively low yields of 18FDG, this remote supply of 18FDG served to demonstrate its unique properties and its utility as a scientific tool for basic research and clinical diagnosis. In the next few years BNL supplied 18FDG to the Hospital of the University of Pennsylvania and also to the National Institutes of Health. Soon, however, most of the major institutions having a cyclotron produced 18FDG for their own use. It is remarkable that 25 years later, the production of 18 FDG at regional cyclotron-synthesis centers and its distribution to remote hospitals and other institutions for clinical use particularly in cancer is the major mode for supplying FDG. In this chapter we will highlight the major milestones in chemistry from the conceptual design through the 18T evolution of its chemical syntheses. We note that there have been other reviews of various aspects of FDG design and chemistry (Fowler & Wolf, 1986) including a very recent article on 18FDG chemistry (BeuthienBaumann et al., 2000).

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Figure 1. (clockwise from upper left) Synthesis of 18FDG for the first human study (left to right, Tatsuo Ido, C-N Wan and Alfred P. Wolf); Delivery of 18FDG to Philadelphia Airport (Tatsuo Ido and Vito Casella); 18FDG injection and imaging in Mark IV scanner (Martin Reivich and Joel Greenberg); Brain images

DESIGN OF 18FDG: THE IMPORTANCE OF C-2 18 FDG was modeled after carbon-14 labeled 2-deoxyglucose (14C-2DG). 2-Deoxy-D-glucose (2-DG) is a derivative of glucose in which the hydroxyl group (-OH) on C-2 is replaced by a hydrogen atom (Figure 2).

D-glucose

2-deoxy-D-glucose

2-deoxy-2-fluoro-D-glucose

Figure 2. Structure of glucose, 2-deoxy-D-glucose (2-DG) and 2-deoxy-2-fluoro-D-glucose (FDG) showing modifications at C-2.

DESIGN AND SYNTHESIS OF 2-DEOXY-2-[ 18 F]FLUORO-D-GLUcosE ( 18 FDG)

309

The biological behavior of 2-DG is remarkably similar to glucose, with a few important differences. Like glucose, 2-DG undergoes facilitated transport into the brain followed by phosphorylation by hexokinase because the hydroxyl group on C-2 is not a critical element for either of these processes. In contrast to glucose, however, metabolism does not proceed beyond phosphorylation because the hydroxyl group on C-2 is crucial in the next step, phosphohexose isomerase. As a result, 2-deoxy-D-glucose-6-phosphate is trapped in the cell providing a record of metabolism. In essence, removal of the hydroxyl on C-2 isolates the hexokinase reaction. This property of 2-DG was noted in 1954 by Sols and Crane (Sols & Crane, 1954) who remarked: "2-deoxyglucose possesses certain advantages over glucose as a substrate for experimental studies with crude preparations of brain and other tissue hexokinases. The phosphate ester formed from 2-deoxyglucose is not inhibitory and it is not a substrate for either phosphohexose isomerase or glucose-6-phosphate dehydrogenase. Thus, the use of 2-deoxyglucose isolates the hexokinase reaction." The translation of the 14C-2-DG method to humans required that 2-DG be labeled with an isotope which decayed by body penetrating radiation and that the chemical properties of the isotope and its position on the deoxyglucose skeleton would not significantly perturb its biochemical and transport properties. Of course, this could be achieved by isotopic substitution of stable carbon in the 2-deoxyglucose structure with carbon11, and this synthesis was accomplished shortly after the development of 18FDG (MacGregor et al., 1981). However, fluorine-18 was chosen for initial studies both because the C-F bond is a strong bond and because its 110 minute half-life was sufficiently long for transport from Long Island to Philadelphia where the first human studies were carried out on the Mark IV scanner (Kuhl et al., 1977). The design of an F-18 labeled version of 2-deoxyglucose hinged on substituting the F-18 on a carbon atom which would preserve the properties of the parent molecule. The choice of C-2 for the fluorine substitution was an obvious one. C-2, unlike other carbon atoms in the molecule, can be modified without interfering with either facilitated transport required to bring the molecule across the blood-brain-barrier (BBB) or the hexokinase reaction. It was also reasonable to assume that 2-deoxy-2-fluoro-D-glucose (FDG) would not be a substrate for phosphohexoseisomerase. Thus, it was predicted that 2-deoxy-2-[18F]fluoro-D-glucose (18FDG) would be a good substrate for hexokinase and that, with the absence of a hydroxyl group on C-2, the phoshorylated product would be intracellularly trapped at the site of metabolism providing a record of metabolic activity which could be imaged externally (Figure 3), The development of 18FDG was further supported by the fact that FDG had been synthesized in unlabeled form and shown to be a good substrate for hexokinase (Bessell et al., 1972). The importance of substituting the fluorine atom on C-2 is illustrated by the dramatic reduction in affinity for hexokinase with 3-deoxy-3-fluoro-D-glucose and 4-deoxy-4-fluoro-Deiucose (Table 1).

310

HANDBOOK OF RADIOPHARMACEUTICALS

BLOOD

BRAIN HK

-*• GLU-6-P—>- other productsH

H

D-glucose (GLU) HK

H

-HU-14C]-2-DG-6-P V ».

H

14 [U-14 C]-2-deoxy-D-glucose (2-DG)

HK H

18

•^

18

FDG-6-P

X"

H

FDG Blood Brain Barrier

Figure 3. FDG model compared to glucose and 2-DG. Note that replacement of the hydroxyl (-OH) group at C-2 does not alter facilitated transport or phosphorylation by hexokinase (HK) but does prevent metabolism beyond the phosphorylation step. Table 1. Substrate specificities for hexokinase. Note that substitutions at C-2 retain specificity for hexokinase while substitutions on C-3 and C-4 result in increases of more than 100 in Km. Substrate

Hexokinase source

K m (mmol)

reference

D-glucose

Yeast

0.17

2-deoxy-D-glucose

Yeast

0.5910.11

Bessell etal., 1973

2-deoxy-2-fluoro-D-glucose

Yeast

0.1910.03

Bessell etal.. 1973

2-deoxy-2-fluoro-D-glucose

Bovine brain

0.2

Machado De Domenech & Sols, 1980

2-deoxy-2-fluoro-D-mannose

Yeast

0.4110.05

Bessell
3-deoxy-3-fluoro-D-glucose

Yeast

70130

Bessell era!., 1973

4-deoxy-4-fluoro-D-glucose

Yeast

84

Bessell etal.. 1973

!

Bessell era/., 1973

DESIGN AND SYNTHESIS OF 2-DEOXY-2-[18F]FLUORO-D-GLUcosE (18FDG)

311

In order to test the hypothesis that FDG would be a good model for 2-DG, FDG was labeled with C-14 (Ido et al., 1978). Autoradiographic studies with [14C]FDG in the rat gave similar results as those obtained for f 14C]2-DG and phosphorylation by hexokinase also proceeded as predicted (Reivich et al., 1979). These studies formed the groundwork for developing a synthesis for 2-deoxy-2-[!8F]fluoro-D-glucose (18FDG) for studies of brain glucose metabolism in humans. However, 18FDG's unique high uptake in rapidly growing tumors (Som et al., 1980) as a result of enhanced tumor glycolysis (Weber, 1977) coupled with its low body background resulted in a very high signal to noise ratio to detect tumors in the body. The low body background from 18FDG is due in part to the fact that 18FDG which is not phosphorylated by hexokinase is excreted (Gallagher et al., 1978). This contrasts to the behavior of glucose which is not excreted due to resorption from urine to plasma via active transport across the renal tubule. The presence of a hydroxyl group on C-2 which occurs in glucose but not 18FDG is required for active transport (Silverman, 1970). This property of low body background resulting from 18FDG excretion which was not anticipated in the initial design of I8FDG for brain studies has elevated it to the forefront as a tracer for managing the cancer patient (Coleman, 2000). FIRST SYNTHESIS OF 18FDG FOR ANIMAL AND HUMAN STUDIES With I8FDG as a goal, the options for rapid incorporation of F-18 in the C-2 position were assessed. Fortunately, there were two syntheses for unlabeled 2-deoxy-2-fluoro-D-glucose in the chemical literature at the time that I8FDG was being developed. One of these involved the electrophilic fluorination of 3,4,6triacetylglucal with the electrophilic fluorination reagent trifluoromethylhypofluorite (CFsOF) (Adamson et al., 1970) which was used in the synthesis of [14C]FDG (Ido et al., 1978). The other synthetic approach to unlabeled FDG involved the use of potassium bifluoride (KHF2) in a nucleophilic displacement reaction (Pacak et al., 1969). Though neither CF3OF nor KHF2 nor the synthetic schemes was directly applicable to the synthesis of 18FDG, it was likely that elemental fluorine (F2) could be substituted for CF3OF based on initial reports that its reactivity could be controlled in diluted form (Barton, 1976). This approach was successful and the fluorination of 3,4,6-tri-O-acetylglucal with elemental fluorine represented a new synthetic route to unlabeled FDG (Ido et al, 1977). Fortunately the methodology for producing [I8F]F2 by the irradiation of a neon target containing F2 via the 20Ne(d,a)i8F reaction using specially prepared nickel irradiation vessel had already been developed (Lambrecht & Wolf, 1973) and applied to the first synthesis of 5-[I8F]fluorouracil (Fowler et al., 1973). Thus electrophilic fluorination of 3,4,6-tri-O-acetyl-D-glucal with [I8F]F2 produced a 3:1 mixture of the F-18 labeled 1,2-difluoroglucose isomer and the 1,2-difluoro-mannose isomers which were separated by preparative gas chromatography. The 1,2-difluoroglucose isomer was hydrolyzed in HC1 to give 18FDG (Figure 4). The yield was about 8%, the purity was >98% and the synthesis time was about 2 hours.

312

HANDBOOK OF RADIOPHARMACEUTICALS OAc Ac

AcO AcO

18

[ F]F2

OH 18 FDM

Figure 4. Synthesis of 18FDG via fluorination with [18F]labeled elemental fluorine (Ido et at., 1978). Because I8FDG had never been administered to a human subject either in labeled or in unlabeled form, there was no adequate safety data to support administration to humans. The literature at the time had one report of an LD50 for FDG of 600 mg/kg in rats (Bessell et al., 1973). This was not sufficient to support human studies. Therefore toxicity studies were performed in mice and dogs with unlabeled FDG (Som et al., 1980). Doses of FDG were 14.3 mg/kg and 0.72 mg/kg administered intravenously at weekly intervals for 3 weeks for mice and dogs respectively. There was a control group, which was injected with vehicle for each species. Mice were weighed weekly and at the end of 3 weeks they were sacrificed and their organs examined grossly and microscopically. For dogs, baseline, 2-hour, 1 -week and 2-week blood and urine samples and a few CSF samples were obtained for analysis. At the end of 3 weeks the dogs were sacrificed and their internal organs were examined grossly and microscopically. Neither mice nor dogs that received FDG showed any gross or microscopic differences with their respective control groups. These results indicated that the anticipated dose of 1 mg of 18FDG (0.014 mg/kg) could be safely administered to human volunteers. This was a factor of 150 times less than that administered to dogs and 3000 times less than that administered to mice without any evidence of acute or chronic toxicity. Radiation dosimetry was estimated based on the tissue distribution of 18FDG in dogs sacrificed at 60 minutes and at 135 minutes post injection of 18FDG (Gallagher et al., 1977). The target organ in these initial estimates was the bladder which received 289 mrem/mCi (Reivich et al., 1979). These estimates were later refined when human distribution and excretion data became available (Jones et al.. 1982). These developments: the design of 18FDG based on a knowledge of structure-activity relationships; the synthesis of [14C]FDG (Ido et al., 1978); autoradiographic comparison of [I4C]FDG and [ I4 C]2-DG (Reivich et al., 1979); the synthesis of 18FDG (Ido et al., 1978) toxicological studies of FDG (Som et al.. 1980): biodistribution of 18FDG in mice and dogs (Gallagher et al., 1977): and dosimetry calculations (Reivich et al., 1979) all combined to support the first studies in humans.

DESIGN AND SYNTHESIS OF 2-DEOXY-2-[ l8 F]FLUORO-D-GLUCOSE ( 1 8 FDG)

313

IMPROVEMENTS AND A MAJOR MILESTONE (1976-1986) During the next 10 years after the development of the electrophilic route to 18FDG, its utility as a radiotracer in the neurosciences and in the diagnosis of heart disease and cancer grew, This stimulated the investigations of different synthetic methods to improve yields thereby to increase availability. Other electrophilic routes were developed and nucieophilic routes were sought (Table 2). The most useful of the electrophilic routes was labeled acetylhypofluorite (CH3CO2[18F] which offered advantages over [I8F]F2 in terms of yield and experimental simplicity. Labeled acetylhypofluorite was readily synthesized via in situ formation in acetic acid or via gas-solid phase synthesis using [18F]F2. However, it was subsequently found that the stereospecificity of acetylhypofluorite was dependent on reaction conditions and solvent with one of the most commonly used methods giving ca. 15% of 2-deoxy-2-[18F]fluoro-D-mannose (18FDM), an isomer with the fluorine atom occupying the axial position. A reinvestigation and analysis of the product distribution from other fsuorination reagents derived from elemental fluorine showed that they all produce the mannose isomer in varying amounts (Bida et al., 1984). The synthesis producing the most acceptable Table 2. Synthetic routes to 18FDG (1976-1986). Substrate [18F]Labeled Precursor Electrophilic Methods 3,4,6-tri-O-acetyl-D-glucal I [18F]F2 [18F]F2 -» CH3CO2(18F]

[ l8 F]F 2 -»CH 3 CO 2 [ l8 F]

D-glucal

[[I8F]F2 -» [18F]XeF2

3 ,4,6-tri-O-acetyl -D-glucal

Nucieophilic Methods Hr 18 F]->Cs( 18 F] Hf I8F] -> Et4N[18F]

H[18F] -> KH[I8F]F2

H[ I8 F] -> K[ 18 F]Kryptofix 2.2.2

Reference

Ido et al., 1978 Shiue et al., 1982; Adam, 1982; Diksic& Jolly^_1983_ Ehrenkaufer et al, 1984; Jewett et al, 1984 Shiue et al, 1.983; Sood et al., 1983

Levy et al., 1982a;Levy et aL, 1982b " Methyl or vinyl 4,6-O-benzylidene-a-D-mannopyranoside- Tewson, 1983a,b; 2,3-cyclic sulfate Tewson & Soderlind, 1985 Szarek et al., 1 ,2-anhydro-3,4:5,6-di-isopropylidene-l -C-nitro-D1982;Beeley et mannitol al. 1984 ^ . ! Hamacher et l,2,4,6-tetra-O-acetyl-2-trifluoromethanesulfonyl-$-Dal, 1986 mannopyranose Methyl-4,6-O-benzylidene-3~O-methyl-2-Otrifluoromethanesulfonyl-p-D-mannopyranoside

314

HANDBOOK OF RADiopHARMACbimcALS

product purity involved the gaseous CH3CO2[18F] fluorination of 3,4,6-tri-O-acetyl-D-glucal in freon-11. In this synthesis, the ratio of 18FDG:18FDM was 95:5. Though the effect of using a mixture of 18FDG and 18 FDM on glucose metabolic rate in the human brain has been reported to be negligible, the use of a mixture was less than ideal because the rate constants and the lumped constants for these two molecules could differ in a non-predictable fashion introducing a variable in human studies. A kinetic comparison of 18FDG and 18 FDM in the rhesus monkey indicates that there is a 20% reduction in apparent cerebral metabolic rates for glucose when 18FDM is used. If this is similar in humans, it was estimated that a 15% impurity of 18FDM would lead to an underestimation of 3% in glucose metabolic rate (Braun el al., 1994). In addition to the production of the F-18 labeled mannose isomer, there were other limitations to the electrophilic route to 18FDG. The nuclear reaction commonly used to produce [18F]F2 was the 20Ne(d,a)18F reaction in a high pressure neon gas target to which a small amount of F2 gas was added (Casella et al., 1980). The targetry to produce [18F]F2 and its maintenance at the time was cumbersome and handling elemental fluorine, the most reactive of all elements, required special precautions. However, the major limitation was that under the best circumstances, only 50% of the label is incorporated into the product. This is also the case in the use of CH3CO2[18F] because half of the label is lost in the conversion of [18F]F2 to CH3CO2[18F]. In terms of F-18 yield, another nuclear reaction the l8O(p,n)18F reaction, was far superior as can be seen when the cross sections are compared (Figure 5) (Ruth & Wolf, 1979). The adaptation of the 18O(p,n)18F reaction to a practical production method which would conserve the inventory of costly and occasionally rare O-18 enriched water stimulated the development of small volume enriched water targets which produced F18 as [ l8 F]fluoride in high yield (Wieland et al., 1986; Kilbourn et al., 1984). Methods for recovering O-18 enriched water for reuse have been reported including the use of an anion exchange resin (Dowex 1 x 1 0 ) which permits a 95% recovery of [18F]fluoride ion and a loss of 18O-enriched water of less than 5 pi from a volume of 3 ml (Schlyer et al., 1990). With the availability of high yields of [18F]fluoride, the development of a high yield nucleophilic route to 18FDG became even more compelling. A number of approaches were reported prior to 1986 (Table 2). All of these were plagued with difficult steps including low incorporation of F-18 and difficulty in removing protective groups. Thus the electrophilic route, with its limitations, remained the method of choice through 1985. A major advance in the synthesis of 18FDG from [18F]fluoride was reported in 1986 when it was discovered that kryptofix [2.2.2] could be used to increase the reactivity of [ l8 F]fluoride (Hamacher et al., 1986). In essence, kryptofix masks the potassium ion which is the counterion of the [ l8 F]fluoride. The reaction of [18F]fluoride with l,3,4,6-tetra-O-acetyl-2-O-trifluoromethanesulfonyl-p-D-manno-pyranose to give

DESIGN AND SYNTHESIS OF 2-DEOXY-2-[ l8 F]FLUORO-D-GLUCOSE ( I8 FDG)

315

300 0

18

O(p,n)18F

20

Ne(d,a)18F

0

5

10

15

20

25

particle energy (MeV) Figure 5. Comparison of fluorine-18 yields from the 20Ne(d,a)18F reaction and the 18O(p,n)18F reaction (Casella et al., 1980; Ruth & Wolf, 1979). l,3,4,6-tetra-O-acetyl-2-[18F]fluoro-p-D-gluco-pyranose gives a 95% incorporation of F-18 and the overall synthesis including purification proceeds in about 60% yield. The synthesis involves 2 steps, displacement with [18F]fluoride and deprotection with HC1 (Figure 6). This was an almost perfect solution to the need to produce 18FDG in high yield and in high purity. It also produced

18

FDG in no-carrier-added form and later

ion chromatographic analysis of various preparations from this route showed the presence of FDG in a mass of 1-40 pg (Alexoff et al., 1992). Thus, this new method served an increasing need in the nuclear medicine and the neuroscience communities which were discovering new uses for

18

FDG. It is also simple and

amenable to automation and, in the 15 years since it was reported, a number of automated synthesis modules have become commercially available (Satyamurthy et al., 1999).

HANDBOOK OF RADIOPHARMACELJTICALS H

18

F 18

FDG Figure 6. Synthesis of FDG via fluorination with [ F]fluoride ion (Hamacher et al., 1986). 18

18

18

FDG SYNTHESIS (1986-PRESENT)

No major new developments have been made following this simple, high-yield nucleophilic route. However, a number of variants have been investigated to improve the displacement and the deprotection steps and considerable effort has been put into fine-tuning the reaction and to identifying impurities and contaminants which are carried through to the final product. This has become more critical with the increasing use of 18

FDG in clinical practice where a pharmaceutical quality product is required.

One of the goals has been to optimize the removal of kryptofix 2.2.2 which is used to facilitate the displacement reaction. Methods have been reported for both the removal (Moerlein et al., 1989; Alexoff et al., 1991) and the detection (Ferrieri et al., 1993; Chaly & Dahl, 1989) of kryptofix. The simplest method to remove kryptofix is the incorporation of a short cation exchange resin in the synthesis system so that the hydrolysate (HC1) passes through the cartridge before final purification (Alexoff et al., 1991). Alternatives to the use of kryptofix 2.2.2 have been investigated in order to avoid its appearance as a contaminant in the final product. These include the use of tetrabutylammonium as the counterion (Yuasa et al., 1997; Brodack et al., 1988) as well as the development of a resin-supported form of [ l8 F]fluoride for oncolumn fluorination (Toorongian et al., 1990). The latter method is synergistic with the use of an anion exchange resin to recover O-18 enriched water for reuse. Several kinds of polymer supported quaternary ammonium and phosphonium salts such as dimethylaminopyridinium or tributylphosphonium have been systematically examined for the on-column synthesis of 18FDG (Ohsaki et al., 1998). Alternatives to deprotection with HC1 have also been investigated. The use of a cation exchange resin was investigated and reported to efficiently hydrolyze the acetylated labeled precursor in 10-15 minutes at 100 degrees thereby eliminating the need for a neutralization step in the synthesis (Mulholland, 1995) and also serving to remove kryptofix 2.2.2. The use of base hydrolysis in the deprotection step has also been investigated as an approach to reduce the need for high temperatures and to decrease the synthesis time. Though epimerization at C-2 is a known reaction of aldoses under basic conditions and in this case would produce

18

FDM as a radiochemical impurity (Varelis & Barnes, 1996), a systematic study of the reaction

conditions for basic hydrolysis determined that epimerization could be limited to 0.5% using 0.33 M sodium hydroxide below 40 degrees for about 5 minutes to avoid the neutralization step in the synthesis (Meyer et al., 1999). 2-Deoxy-2-chloro-D-glucose

(C1DG) was identified

determination of the specific activity of

18

as an impurity during ion chromatographic

FDG preparations from the nucleophilic route (Alexoff et al..

DESIGN AND SYNTHESIS OF 2-DEOXY-2-[ l8 F]FLUORO-D-GLUcosE ( 18 FDG) 1992).

517

C1DG is produced as a competing displacement reaction with chloride ion which comes from

different sources including HC1 used in the hydrolysis step. In typical 18FDG preparations, C1DG is present in a total amount of <100 pg as determined by ion chromatography and pulsed amperometric detection. Larger amounts are produced when larger amounts of the triflate precursor are used. The amount of C1DG can be reduced by using sulfuric acid instead of HC1 for hydrolysis. The reduction of the amount of C1DG has also been an impetus for avoiding HC1 in the hydrolysis step. Though C1DG does not present a toxicity problem, its presence is not desireable from the standpoint of pharmaceutical quality. OUTLOOK Advances in chemistry and the remarkable properties of 18FDG have largely overcome the limitations of the 110 minute half-life of fluorine-18 so that 18FDG is now available to most regions of the US from a number of central production sites. This avoids the need for an on-site cyclotron and chemistry laboratory and has opened up the use of 18FDG to institutions which have a PET scanner (or other imaging device) but no cyclotron or chemistry infrastructure. Currently 18FDG is used by many hospitals as an "off the shelf radiopharmaceutical for clinical diagnosis in heart disease, in seizure disorders and in oncology, the area of most rapid growth. However, its ready availability has opened the possibility to also use it in more widespread applications in the human neurosciences including drug research and development (Fowler et al., 1999). This is an important application because with 18FDG it is possible to determine which brain regions are most sensitive to the effects of a given drug. Because glucose metabolism reflects, in part, the energy involved in restoration of membrane potentials, regional patterns may be used to generate hypotheses as to which molecular targets are mediating the effects of the drug. Also a baseline study can be run allowing intra-subject comparison before and after the drug. Since subjects are awake and alert at the time of the study, the behavioral and therapeutic effects of the drug and their association with metabolic effects can be measured. Though the use of a functional tracer like 18FDG is not as precise as the use of a radiotracer which is more specific for a given neurotransmitter system, it nonetheless provides a measure of the final consequences of the effects of the drug on the human brain. This is important because even though a drug may interact with a particular neurotransmitter, it may be the downstream consequences of that interaction which are of relevance to its pharmacological effects. When radiotracer availability permits, the ideal situation is to pair an 18FDG measurement with a neurotransmitter specific measurement and in that way to correlate neurotransmitter-specific effects with regional metabolic effects. 18FDG has many advantages as a scientific tool for preclinical studies in small animals when it is coupled with small animal imaging devices. Because the 18FDG method requires a ~30 minute uptake period before the imaging is actually done the animals can be awake during this period and anesthetized immediately before imaging, thus avoiding the effect of anesthesia on the behavior of the tracer. ACKNOWLEDGMENT This research was performed in part at Brookhaven National Laboratory under contract DE-AC0298CH10886 with the U.S. Department of energy and was supported by its Office of Biological and Environmental Research. The authors also thank David Schlyer for his valuable comments.

318

HANDBOOK OF RADIOPHARMACEUTICALS

REFERENCES Adam MJ (1982) A rapid, stereoselective, high yielding synthesis of 2-deoxy-2-fluoro-D-hexopyranoses: Reaction of glycols with acetyl hypofluorite. J. Chem. Soc. Chem. Commun. 730–732. Adamson J, Foster AB, Hall LD, Johnson RN and Hesse RH. (1970) Fluorinated carbohydrates. Part IE. 2Deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose. Carbohydrate Res., 15, 351–359. Alexoff DL, Casati R, Fowler JS, Wolf AP, Shea C, Schlyer DJ and Shiue C-Y (1992) Ion chromatographic analysis of high specific activity 18FDG preparations and detection of the chemical impurity in 2deoxy-2-chloro-D-glucose. Appl. Radiat. Isot., 43, 1313–1322. Alexoff DL, Fowler JS and Gatley SJ (1991) Removal of the 2.2.2 cryptands (kryptofix 2.2.2) from 18FDG by cation exchange. Appl. Radiat. I sot., 42, 1189–1193. Barton DHR, Hesse RH, Markwell RE, Pechet MM and Rozen S (1976) Fluorination at saturated carbon. 2. Direct fluorination of steroids. J. Am. Chem. Soc., 98, 3036-3037. Beeley PA, Szarek WA, Hay GW and Perlmutter MM (1984) A synthesis of 2-deoxy-2-[18F]fluoro-Dglucose using accelerator-produced 18F-fluoride ion generated in a water target. Can. J. Chem., 62, 2709–2711. Bessell EM, Foster AB and Westwood JH (1972) The use of deoxyfluoro-D-glucopyranoses and related compounds in a study of yeast hexokinase specificity. Biochem. J., 128, 199–204. Bessell EM, Courtenay VD, Foster AB, Jones M and Westwood JH (1973) Some in vivo and in vitro antitumor effects of the deoxyfluoro-D-gluco-pyranoses. Eur. J. Cancer., 9, 463–470. Beuthien-Baumann B, Hamacher K, Oberdorfer F and Steinbach J (2000) Preparation of fluorine-18 labelled sugars and derivatives and their application as tracer for positron-emission-tomography. Carbohydrate Res., 327, 107–118. Bida GT, Satyamurthy N and Barrio J (1984) The synthesis of 2-[18F]fluoro-2-deoxy-D-glucose using glycals: a reexamination. J. Nucl. Med., 25, 1327–1334. Braun AR, Carson RE, Adams HR, Finn RD, Francis BE and Herscovitch P (1994) A kinetic comparison of [l8F]2-fluoro-2-deoxyglucose and [18F]2-fluoro-2-deoxymannose using positron emission tomography. Nucl. Med. Biol., 6, 857–63. Brodack JW, Dence CS, Kilbourn MR and Welch MJ (1988) Robotic production of 2-deoxy-2-[18F]fluoro-Dglucose: a routine method of synthesis using tetrabutylammonium [l8F]fluoride. Appl. Radiat. Isot., 39, 699-703. Casella V, Ido T, Wolf AP, Fowler JS, MacGregor RR and Ruth TR (1980) Anhydrous F-18 labeled elemental fluorine for radiopharmaceutical preparation. J. Nucl. Med., 21, 750-757. Chaly T and Dahl JR (1989) Thin layer chromatographic detection of kryptofix 2.2.2 in the routine synthesis of [18F]2-fluoro-2-deoxy-D-glucose. Nucl. Med. Biol., 16, 385-387. Coleman RE (2000) FDG imaging. Nucl. Med. Biology, 27, 689-690. Diksic M and Jolly D (1983) New high-yield synthesis of l8F-labeled 2-deoxy-2-fluoro-D-glucose. Int. J. Appl. Radiat. Isot., 34, 893–896. Ehrenkaufer RE, Potocki JF, and Jewett DM (1984) Simple synthesis of F-18-labeled 2-fluoro-2-deoxy-Dglucose: concise communication. J. Nucl. Med., 25, 333–337. Ferrieri RA, Schlyer DJ, Alexoff DL, Fowler JS and Wolf AP (1993) Direct analysis of kryptofix 2.2.2 in 18 FDG by gas chromatography using a nitrogen-selective detector. Nucl. Med. Biol., 20, 367–369.

DESIGN AND SYNTHESIS OF 2-DEOXY-2-[18F]FLUORO-D-GLUcosE ( 18 FDG)

319

Fowler JS, Finn RD, Lambrecht RM and Wolf AP (1973) The synthesis of 5-fluorouracil-18F. J, Nucl. Med., 14, 63-64. Fowler JS and Wolf AP (1986) 2-Deoxy-2-[18F]fluoro-D-glucose for metabolic studies: current status. Appl. Radial, Isot., 37, 663–668. Fowler JS, Volkow ND, Wang G-J, Ding Y-S and Dewey S (1999) PET and drug research and development. J. Nucl. Med., 40, 1154–1163. Gallagher BM, Ansari A, Atkins H, Casella V, Christman DR, Fowler JS, Ido T, MacGregor RR, Som P, Wan C-N, Wolf AP, Kuhl DE and Reivich M (1977) Radiopharmaceuticals XXVII: 18F-Labeled 2deoxy-2-fluoro-D-glucose as a radiopharmaceutical for measuring regional myocardial glucose metabolism in vivo: tissue distribution and imaging studies in animals. J. Nucl. Med., 19, 990–997. Gallagher BM, Fowler JS, Gutterson NI, MacGregor RR, Wan C-N and Wolf AP (1978) Metabolic trapping as a principle of radiopharmaceutical design: some factors responsible for the biodistribution of [18F]2-deoxy-2-fluoro-D-glucose. J. Nucl. Med., 19, 1154–1161. Hamacher K, Coenen HH and Stocklin G (1986) Efficient stereospecific synthesis of NCA 2-[18F]fluoro-2deoxy-D-glucose using aminopolyether supported nucleophilic substitution. J. Nucl. Med., 27, 235238. Ido T, Wan C-N, Casella V, Fowler JS, Wolf AP, Reivich M and Kuhl DE (1978) Labeled 2-deoxy-Dglucose analogs, 18F-labeled 2-deoxy-2-fluoro-D-glucose, 2-deoxy-2-fluoro-D-mannose and l4C-2deoxy-2-fluoro-glucose. J. Labelled Cpd. and Radiopharm., 14, 171-183. Ido T, Wan C-N, Fowler JS and Wolf AP (1977) Fluorination with F2. A convenient synthesis of 2-deoxy-2fluoro-D-glucose. J. Org. Chem., 42, 2341–2342. Jewett DM, Potocki JF and Ehrenkaufer RE (1984) A gas-solid phase microchemical method for the synthesis of acetyl hypofluorite J. Fluorine Chem., 24, 477–484. Jones SC, Alavi A, Christman D, Montanez I, Wolf AP and Reivich M (1982) The radiation dosimetry of [F18]-2-fluoro-2-deoxy-D-glucose in man. J. Nucl. Med., 23, 613–617. Kilboum MR, Hood JT and Welch MJ (1984) A simple 18O-water target for 18F production. Int. J. Appl. Radiat. Isot., 35, 599-602. Kuhl DE, Hoffman EJ, Phelps ME, Ricci AR and Reivich M (1977) Design and application of the Mark IV scanning system for radionuclide computed tomography of the brain. In Medical Radionudide Imaging, Vol I, International Atomic Energy Agency Symposium on Medical Radionuclide Imaging, Los Angeles, CA, Oct 25–29, 1976, IAEA, Vienna, 309-320. Lambrecht R and Wolf AP (1973) Cyclotron and short-lived halogen isotopes for radiopharmaceutical applications. In Radiopharmaceuticals and Labeled Compounds, Volume 1, International Atomic Energy Agency, Vienna, 275-290. Levy S, Elmaleh D and Livni E (1982a) A new method using anhydrous [18F]fluoride to radiolabel 2[18F]fluoro-2-deoxy-D-glucose. J. Nucl. Med., 23, 918-922 Levy S, Livni E, Elmaleh D and Curatolo WJ (1982b) Direct displacement with anhydrous fluoride of the C2 trifluoromethanesulfonate of methyl 4,6-O-benzyh'dene-3-O-mediyl-2-O-trifluoromethylsulphonyl-p-Dmannoside. J. Chem. Soc. Chem. Comm., 972-973. MacGregor RR, Fowler JS, Wolf AP, Shiue C-Y, Lade RE and Wan C-N (1981) A synthesis of 11C-2deoxy-D-glucose for regional metabolic studies. J. Nucl. Med., 22, 800–803.

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Machado De Domenech EE and Sols A (1980) Specificity of hexokinases towards some uncommon substrates and inhibitors. FEBS Lett., 119, 174–176. Meyer G-J, Matzke KH, Hamacher K, Fuchtner F, Steinbach J, Notohamiprodjo G and Zilstra S (1999) The stability of 2-[18F]fluoro-deoxy-D-glucose towards epimerization under alkaline conditions. Appl. Radiat. Isot., 51, 37–41. Moerlein SM, Brodack JW, Siegel BA and Welch MJ (1989) Elimination of contaminant kryptofix 2.2.2 in the routine production of 2-[18F]fluoro-2-deoxy-D-glucose. Appl. Radiat. hot., 40, 741 -743. Mulholland GK (1995) Simple rapid hydrolysis of acetyl protecting groups in the FDG synthesis using cation exchange resins. Nucl. Med. Biol., 22, 19–23. Ohsaki K, Endo Y, Yamazaki S, Tomoi M and Iwata R (1998) Polymer-supported catalysts for on-column preparation of 2-deoxy-2-[18F]fluoro-D-glucose. Appl. Radiat. Isot., 49, 373-378. Pacak J, Tocik Z and Cerny M (1969) Synthesis of 2-deoxy-2-fluoro-D-glucose. J. Chem. Soc. Chem. Comm., 77. Reivich M, Kuhl D, Wolf A, Greenberg J, Phelps M, Ido T, Casella V, Fowler J, Hoffman E, Alavi A. Som P and Sokoloff L (1979) The [18F]fluorodeoxyglucose method for the measurement of local cerebral glucose utilization in man. Circ. Res., 44, 127–137. Ruth TJ and Wolf AP (1979) Absolute cross sections for the production of 18F via the l8O(p,n)18F reaction. Radiochim. Acta., 26, 21–24. Satyamurthy N, Phelps ME and Barrio JR (1999) Electronic generators for the production of positron-emitter labeled radiopharmaceuticals; Where would PET be without them? Clinical Positron Imaging, 2, 233-252. Schlyer DJ, Bastos MAY, Alexoff A and Wolf AP (1990) Separation of [ l8 F]fluoride from [l8O]water using anion exchange resin. Appl. Radiat. Isot., 41, 531 –533. Shiue C-Y, Salvadori PA, Wolf AP, Fowler JS and MacGregor RR (1982) A new improved synthesis of 2deoxy-2-[18F]fluoro-D-glucose from l8F-labeled acetyl hypofluorite. J. Nucl. Med., 23, 899–903. Shiue C-Y, To KC, and Wolf AP (1983) A rapid synthesis of 2-deoxy-2-fluoro-D-glucose from xenon difluoride suitable for labeling with 18F. J Labelled Cpd. Radiopharm., 20, 157-162. Silverman M (1970) Specificity of monosaccharide transport in the dog kidney. Am J Physiology, 218, 743750. Sokoloff L (1979) Mapping of local cerebral functional activity by measurement of local cerebral glucose utilization with [14C]deoxyglucose. Brain, 102, 653-668. Sols A and Crane RA (1954) Substrate specificity of brain hexokinase. J. Biol. Chem., 210, 581–595. Som P, Atkins HL, Bandoypadhyay D, Fowler JS, MacGregor RR, Matsui K, Oster ZH, Sacker DF, Shiue CY, Turner H, Wan C-N, Wolf AP and Zabinski SV (1980) A fluorinated glucose analog, 2-fluoro-2deoxy-D-glucose (F-l 8): nontoxic tracer for rapid tumor detection. J. Nucl. Med., 21, 670–675. Sood S, Firnau G, and Gamett ES (1983) Radiofluorination with xenon difluoride. Int. J. Appl. Radiat. Isot., 34, 743–745 Szarek W, Hay GW, and Perlmutter MM (1982) A rapid stereospecific synthesis of 2-deoxy-2-fluoro-Dglucose using fluoride ion. J. Chem. Soc. Chem. Comm., 1253–1254.

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Tewson TJ (1983a) Cyclic sulfur esters as substrates for nucleophilic substitution. A new synthesis of 2deoxy-2-fluoro-D-glucose. J. Org. Chem., 48, 3507-3510. Tewson TJ (1983b) Synthesis of no-carrier-added fluorine-182-fluoro-2-deoxy-D-glucose. J. Nucl. Med,, 24, 718–721. Tewson TJ and Soderlind M (1985) I-Propenyl-4,6-O-benzylidene-p~mannopyranoside-2,3-cyclic sulfate: a new substrate for the synthesis of [F-18]-2-deoxy-2~fluoroglucose. J. Nucl. Med., 26, PI29 (abstract). Toorongian SA, Mulholland GK, Jewett DM, Bachelor MA and Kilboum MR (1990) Routine production of 2-deoxy-2-[18F]fluoro-D-glucose by direct nucleophilic exchange on a quaternary 4-aminopyridinium resin. Int. J. Had. Appl. Instrum. B., 17, 273-279. Varelis P and Barnes RK (1996) Epimerization of 2-deoxy-2-[18F]fluoro-D-glucose under basic conditions. A convenient method for the preparation of 2-deoxy-2-[18F]fluoro-D-mannose. Appl. Radiat. hot., 47, 731–733. Weber G (1977) Enzymology of cancer cells. N.Eng.J. Med., 296, 541–551. Wieland BW, Hendry GO, Schmidt DG, Bida G and Ruth TJ (1986) Efficient small-volume l8O-water targets for producing 18F fluoride with low energy protons. J. Labelled Cpd Radiopharm., 23, 12051207. Yuasa M, Yoshida H and Hara T (1997) Computer-controlled synthesis of [18F]FDG by the'tetrabutylammonium method: achievement of high yield, purity, reproducibility, reliability, and safety. Appl. Radiat. hot., 48, 201–205.

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10. TECHNETIUM RADIOPHARMACEUTICALS ASHFAQ MAHMOOD AND ALUN G. JONES

Department of Radiology, Brigham and Women's Hospital and Harvard Medical School, 220 Longwood Avenue, Boston MA 02115 USA

INTRODUCTION The existence among the second row transition metals of the element with atomic number 43 was predicted by Mendeleev in 1869 and first discovered by Perrier and Segre in 1937 (Perrier & Segre, 1937). Seven years later Segrd with the help of a classical scholar gave the element the name technetium, derived from the Greek word technetos meaning artificial. Today twenty-five isotopes and ten isomers of this man-made element, have been identified, formally ranging from 88 to 113 atomic mass units. While the most common of these nuclides is technetium-99 (P~, E = 293 keV; t i/2 = 2.13 x 105 y) by virtue of its being a major byproduct of uranium-235 fission, the most widely used isotope is technetium-99m (y, E = 140.5 keV; t 1/2 = 6.01 h). This metastable state, discovered by Segre and Seaborg in 1937 (Segre & Seaborg, 1938) is a product of molybdenum-99 decay (t1/2 = 65.94 h) and is currently the most commonly used isotope in nuclear medical imaging. Two other radionuclides of technetium have also been employed to some extent in research: technetium-95m (t1/2 = 61 d), a long-lived tracer that is occasionally used in pharmacokinetic studies; and technetmm-94m (t1/2 = 52 min), a positron emitter that is used in animal studies to take advantage of the quantitative capability offered by PET. The widespread use of technetium-99m in radiopharmaceuticals is due to several factors. The most important of these is the favorable mode of decay which includes a highly abundant 140 keV gamma ray that can be very efficiently detected with the thallium-doped sodium iodide crystals found in Anger cameras. The isotope has a short half-life of 6.01 hours, and is accompanied by a relatively low level of nonpenetrating radiation, i.e., conversion electrons; these characteristics result in a generally favorable dosimetry for the patient. The half-life also makes temporally possible the chemical manipulations necessary for preparing conveniently a range of radiopharmaceuticals. In addition, the efficient separation of high-specific-activity samples of the radionuclide from its parent using the 99Mo99mTc generator system, first developed at the Brookhaven National Laboratory in 1958 by Tucker and Green (Tucker et al., 1958) and later elaborated by Richards (Richards, 1966; Richards et al., 1982), is a great convenience. The concentration of technetium (including both 99mTc and 99Tc) eluted as pertechnetate ion from the generator is strongly dependent on its elution history but is generally in the range of 1017 to 10-10 M. This precludes characterization of its metal complexes at no-carrier-added concentrations beyond electrophoretic and chromatographic profiling with thin-layer chromatography or HPLC coupled with gamma detection. However, over the years, the availability and use (with appropriate precautions) of macroscopic

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S, Redvanly. ©2003 John Wiley & Sons, Ltd

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concentrations of the long-lived technetium-99 isotope has enabled the use of standard methods of chemical characterization to determine the exact nature and three-dimensional structure of these molecules. Along with the use of rhenium, the third row transition-metal congener of technetium, this use of technetium-99 has allowed the elucidation of the underlying chemistry, structure, and other physiochemical parameters of technetium radiopharmaceuticals. CHEMISTRY The chemistry of most technetium-based radiopharmaceuticals begins with an aqueous solution of sodium pertechnetate (Na99mTcO4), the form in which technetium is available when eluted from the generator. This +7 oxidation state species does not readily form complexes with donor ligands or chelates and, therefore, must be reduced to a lower oxidation state. The only common exception to this is technetium heptasulfide (Tc2S7), which is thought to be present in the agent used to image the reticuloendothelial system of the liver, spleen, and bone marrow. Reduction of pertechnetate can be accomplished with a number of agents, the most common of which are stannous chloride and hydrochloric acid; others include sodium dithionite, sodium borohydride, ascorbic acid, hydrazine, and tertiary phosphines. Upon reduction in the presence of coordinating ligands or chelates, the technetium may attain a lower oxidation state from (I) to (VI) depending on the denticity and donating ability of the coordinating ligand(s). This variation in oxidation state together with the range of structural geometries that may be attained with coordination numbers ranging from 4 to 7 affords technetium a rich chemistry that has been exploited to synthesize a wide range of complexes. The reader is directed to a number of excellent reviews on the subject (Horn & Katzenellenbogen, 1997a; Liu el al., 1997; Jurisson & Lydon, 1999; Lister-James & Dean, 1999; Liu & Edwards, 1999; Bandoli et al.. 2001). In general, technetium-containing complexes are formed by one of two reaction sequences. The first is a single-step reaction in which generator-eluted TcO4 is added to a sterile vial containing both the coordinating ligand(s) and the reducing agent followed by incubation of the vial at ambient or elevated temperature for an appropriate time. The second is a two-step reaction in which the TcO4 is first reduced in the presence of a labile ligand that stabilizes the metal in an appropriate oxidation state, followed by addition of this intermediate complex to a vial containing the final ligand(s) which are stronger donors and have greater thermodynamic stability. The transfer of the prereduced metal to the desired donor ligand(s) forms the final metal complex and the process is referred to as "transmetalation" or "exchange labeling". RADIOPHARMACEUTICALS The first application of technetium as a radiopharmaceutical in the clinic was at the University of Chicago where Harper used 99mTcO4 eluted from a 99Mo-99mTc generator developed at Brookhaven, to obtain images of the liver, brain, and thyroid (Harper et al., 1964a,b). Subsequently an "instant kit" was developed by Atkins, Eckelman and Richards (Richards & Atkins, 1968; Eckelman & Richards, 1970; Eckelman & Richards, 1971) in which a single vial containing both a reducing agent (stannous chloride) and a complexing agent produced 99mTc-complexes in high radiochemical yield in sterile aqueous solution. This led to the development of early technetium imaging agents such as 99mTc-DTPA for renal imaging (Eckelman & Richards 1970), "mTc-pyrophosphate for skeletal imaging (Subramanian & McAfee, 1971). technetiumlabeled red blood cells for cardiac ejection fraction and blood volume imaging (Smith & Richards. 1976).

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99m

Tc-MDP for skeletal imaging (Subramanian et al., 1975), 99mTc-HIDA for hepatobiliary imaging (Loberg et al, 1976), and 99mTc-glucoheptonate for renal imaging, almost all of which are still used in clinical nuclear medicine today.

At the time of the development of these agents, the basic chemistry of technetium was less well understood with the result that most of these agents were not fully characterized. Since then, because of the ready availability of the long-lived isotope technetium-99, considerable knowledge of the chemistry of technetium has been acquired, and nowadays most technetium-based radiopharmaceuticals undergo rigorous and complete structural and chemical characterization as an essential component of the approval process required by regulatory agencies such as the FDA.

BRAIN AGENTS Prior to 1980, imaging of the brain in humans with technetium agents was limited to situations where the blood-brain barrier was compromised or damaged. The need for early diagnosis of neurological disorders where this was not the case required that agents penetrate the blood-brain barrier and be retained sufficiently long to provide useful images. This imposed certain criteria that complexes must possess in order to accumulate in the brain, the most important of which are overall charge neutrality, sufficient lipophilicity, and some mechanism of retention. Brain perfusion agents whose distribution reflected regional cerebral blood flow (rCBF) and met these criteria were first introduced with 99mTc-propyleneamine oxime (PNAO) complexes (Troutner et al., 1984). Subsequent development to optimize the in vivo brain uptake and improve the retention in cerebral tissue (Leonard et al., 1986) led to the FDA approval of a Tc(V) complex of hexamethylpropyleneamine oxime (HMPAO or Ceretec™).

The uncomplexed HMPAO chelate itself possess two chiral centers and can be obtained either as a meso- or as a D,L-isomer. Technetium(V) complexes of both isomers have been investigated and, while those formed with the meso-isomer are relatively more stable, those of the D,L-isomer provide good rCBF images. This relative "instability" of D,L-99mTc-HMPAO compared with the rneso-99mTc-HMPAO isomer has been cited as the reason for the greater propensity of the D,L-99mTc-HMPAO to form charged species once the complex has passed through the blood-brain barrier, thus providing a trapping mechanism and ensuring its retention (Nowotnik et al., 1985; Sharp et al., 1986). Intracerebral interaction with glutathione to produce alternative

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hydrophilic species has also been proposed as a trapping mechanism (Ballinger et al., 1988; Neirinckx et al., 1988). Based upon earlier observations of the affinity of Tc(V) species for thiols as donor ligands (DePamphilis et al., 1978; Smith et al., 1978), tetradentate N2S2 chelates were developed by several research groups (Bums et al., 1981; Davison et al., 1981; Kung et al., 1984). These molecules all contain two nitrogens and two thiols as the donor groups within the chelate framework and form stable square pyramidal mono-oxoTc(V) complexes with the oxygen atom at the apex of the pyramid formed by the four coordinating N2S2 donor groups. Chelates such as these have led to the development of several agents including the identification of the ethyl cysteinate dimer (ECD) and its mono-oxotechnetium(V) complex (Neurolite™) by Cheesman et al. (1988). The ligand itself has two chiral centers and can be synthesized either as an LJL-ECD or as a D,DECD chelate. Both chelates form similar chiral and symmetric square pyramidal Tc(V)oxo complexes which are neutral and lipophilic.

L,L-[TcvOECD]

Their in vivo behavior, however, is quite different with the L,L-TcO-ECD complex displaying a higher brain retention and faster blood clearance than the D,D-TcO-ECD complex. Mechanistically, the stereoselective in vivo behavior of these complexes results from the passage of the neutral molecule across the blood-brain barrier, followed by enzymatic hydrolysis by cerebral esterases of one of the ester groups to an acid functionality. This transforms the neutral L,L-TcO-ECD complex to a charged specie and prevents its egress from the brain, thus providing a trapping mechanism. While both complexes permeate the blood-brain barrier equally well. L,L-TcO-ECD is hydrolyzed more rapidly compared with D,D-TcO-

MRP-12

ECD in the primate and human brain (Walovitch et al., 1989; Walovitch et al., 1991). Ester hydrolysis of L,L-TcO-ECD also occurs in the blood, which results in faster blood clearance primarily through the kidneys, thus providing increasing brain/blood ratios over time (Walovitch et al., 1991). Other brain perfusion agents that have been studied in humans include the Tc-boronic acid adducts of dioximes (Tc-BATO), particularly the 2-methylpropyl derivative (Narra et al., 1990), and the oxo-Tc(V) complex MRP-12 (Bossuyt et al., 1991). The Tc-BATO complexes are synthesized via an elegant "template

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synthesis" procedure developed by researchers at Bristol-Meyers Squibb (Stewart et al., 1990). In a single reaction vial, 2-methylboronic acid, dimethylglyoxime, and stannous chloride are reacted with generatoreluted TcO4 under acidic conditions to produce a neutral seven-coordinate Tc(III)-BATO-2MP complex. While the Tc-BATO-2MP and MRP-12 complexes display moderate brain uptake, their in vivo pharmacokinetics as compared with those of D,L-99mTc-HMPAO or L,L-TcO-ECD have hindered their further development for routine clinical utility. Currently most clinical brain imaging studies with technetium-99m, aimed at the detection of rCBF changes in various pathophysiologies, are performed with D,L-99mTc-HMPAO or L,L-TcO-ECD. Recently, however, developments in designing potential neuroreceptor-specific technetium radiopharmaceuticals have shown great progress. These small receptor-specific agents are unique and may provide a distinct window into specsfic pathophysiologies compared with the brain-perfusion agents. These neuroreceptor-specific molecules are described below along with other technetium-based receptor-mediated agents that seem likely to open new avenues in SPECT imaging. HEART AGENTS Routine myocardial perfusion imaging in nuclear medicine came into being with the advent of [201Tl]thallous chloride. The ionic radius of 20IT1+ is slightly larger than that of K+ and it is actively transported into myocytes, at least partially by the Na+/K+ pump (Pitt & Strauss, 1976; Atkins et al., 1977). While thallium201 is relatively efficiently extracted by myocytes from the blood, its long half-life and imaging characteristics have led to the search for technetium perfusion agents. The first of these agents, the cationic Tc(III)bis-arsine, [TcBr2(diars)2]+, and Tc(III)bis-phosphine, [TcCl2(dmpe)2]+, complexes were described by Deutsch et al. (1981). Although the complexes produced satisfactory images in dogs and rabbits, in vivo reduction to a neutral Tc(II) specie in humans inhibited adequate accumulation in the human myocardium (Gersonetal., 1984).

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©

[Tc(dmpe)3]+

[TcCI2(dmpe)2]

©

©

This work stimulated research into other types of cationic species, for example, the technetium(I) complexes including [Tc(dmpe)3]+ (Deutsch et al., 1989), [Tc(tmp)6]+ (Gerundini et al., 1986), and [Tc(arene)2]+ (Wester et al., 1991). All, however, show slow clearance due to their high binding to protein in the blood. Some of these complexes represent the first examples among technetium species where biodistribution in the target tissue, in this case the heart, has been found to be markedly dissimilar in man to that in the higher primates and other species. The first complex to image successfully human myocardial tissue is the hexakis tert-butylisonitrile technetium(I) complex, [Tc(TBI)6]+, reported by Abrams et al. (1983). Although [Tc(TBI)6]+ is easily synthesized, quite stable, and efficiently extracted into myocardial tissue, the large tert-butyl groups impart substantial lipophilicity to the complex, resulting in high lung and liver uptake and necessitating delay of an hour to allow activity to decrease in these nontarget organs before adequate cardiac images can be obtained (Holman et al., 1984). Blood clearance, by contrast, is rapid because these complexes show little protein binding in blood. Further research to achieve more optimal in vivo distribution of this class of complexes has led to the development of hexakis(2-methoxyisobutylisonitrile)Tc(I), [Tc(I)(MIBI)6]+ (Cardiolite™ or sestamibi), an FDA-approved agent for clinical myocardial perfusion imaging (Taillefer et al., 1988). -,e

X

7\

N

^h [Tc!(TBI)6r

-O

[Tc1(CPI)6]+

Additional functionalized technetium(I) isonitrile complexes have been designed to hydrolyze in vivo in nontarget tissue to improve contrast in the heart image; the most studied of these is [Tc(I)(CPI)6]+ (Holman et

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at., 1987). The ether isonitrile derivatives, such as MIBI, clear primarily through the hepatobilary system. Analysis of bile samples indicates metabolism of the ether groups of the complex (Kronauge et al., 1990). The ester derivatives, such as CPI (Kronauge et al, 1992), also form a series of progressively more hydrophilic metabolites which clear through the renal system and have been identified in the urine. Mechanistic studies using cultured myocytes and isolated perfused hearts indicate the uptake mechanism of these lipophilic cations to be mediated by both the cytoplasmic and, more important, the mitochondrial membrane potentials (Muller et al., 1987; Delmon-Moingeon et al., 1990; Piwnica-Worms et al., 1990), thus facilitating their localization in viable myocardial tissue, skeletal muscle, and in certain types of tumors. Other lipophilic cations that have been approved as myocardial perfusion agents include the rrans-dioxo technetium(V) bisphospine complex known commercially as Tc-tetrofosmin (Higleyet al., 1993) (Myoview™) and the mixed-ligand technetium(III) Schiff-base bisphosphine Tc-QJ2 complexes (Rossetti et al, 1994). Both are derived from the phosphine-based [TcCl2(dmpe)2]+complexes. However, the earlier

©

©

[Tc-tetrofosmin]+

[Tc-Q12]+

problem posed by in vivo reduction which led to inadequate accumulation of [TcCl2(dmpe)2]+ complexes in human myocardial tissue has now been solved. In the case of Tc-tetrofosmin, keeping the concentrations of SnCl2 and the appropriate phosphine low and performing the complexation reaction at room temperature are designed to prevent further reduction to the Tc(III) or Tc(I) specie. For the Q-complexes, this is accomplished by the introduction of the tetradentate Schiff base and the use of a two-step procedure, which entails the initial formation of a Tc(V) intermediate [TcvO(Schiff base)]* by reduction of 99mTcO4 with stannous chloride in the presence of the Schiff base ligand, followed by addition of the tris 3methoxypropylphosphine to the intermediate complex and a concomitant reduction/substitution step to form the final Tc(III) mixed-ligand Tc(III)-Q12 complex. While both of these complexes are structurally quite different from [Tc(I)(MIBI)6]+, both are lipophilic cations that accumulate in myocardial tissue by a mechanism similar to that of [99mTc(I)(MIBI)6]+. More intriguing is the effectiveness of introducing ether groups into the structures of these molecules for this particular application, something that has never been adequately explained. 99m

Tc-teboroxime (Cardiotec™ ) was the first of the neutral technetium complexes evaluated as myocardial perfusion agents (Treher et al., 1989; Stewart et al., 1990). This Tc-BATO complex (similar to those

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described previously) is also prepared using the "template synthesis" procedure in which the sevencoordinate Tc(III) BATO complex is formed by the SnCl2 reduction of 99mTcO4 in the presence of cyclohexanedione dioxime and methylboronic acid under acidic conditions (Linder et al., 1990). In clinical studies the compound displays high myocardial extraction that accurately reflects blood flow but also shows a rapid washout from the heart due to the lack of a retention mechanism. In practice this means that imaging must be performed immediately after administration, but this circumstance also holds out the possibility of repeated studies within a short time-frame, something not possible with the competing agents that have a longer biologic half-life in the heart (Leppo et al., 1991).

Tc-Teboroxime

Tc-NOET

Another neutral technetium(V) nitrido complex, Tc-NOET, has been identified by Pasqualini et al. (1994) and evaluated as a myocardial perfusion agent. While the mechanism of myocardial uptake is not certain, subcellular studies suggest that the complex is localized predominantly in the cell-membrane fraction (Uccelli et al., 1995). Initial human studies indicate that the heart uptake is correlated with the coronary blood flow but that redistribution occurs after the initial uptake. Furthermore, high lung and liver levels coupled with a slow blood clearance because of binding to erythrocytes has hampered its routine use in myocardial perfusion imaging (Johnson et al., 1997).

AGENTS FOR HYPOXIA Myocardial infarction, ischemia, and stroke may result in a condition of hypoxia where, due to reduced perfusion, the oxygen content of the tissue becomes lowered. Certain tumors which outgrow their blood supply can also become hypoxic, thus affecting their response to radiotherapy and chemotherapy (Brizel et al., 1996; Hockel et al., 1996). While 18F-labeled 2-nitroimidazoles were designed to image hypoxic tissue with PET (Grierson et al, 1989), recent reports demonstrating 99mTc-based imaging agents for hypoxic tissue are also encouraging. Arribng these are the nitroimidazole derivatives BSM-181321 and BRU-59-2 based on the 99mTc-propyleneamine oxime (PNAO) complexes (Di Rocco et al., 1997; Johnson et al., 1998). Both

BMS-181321

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331

complexes contain the 2-nitroimidazole functionality and have been shown in vivo to localize in ischemic tissue with BRU-59-2 displaying better overall in vivo uptake and pharmacokinetics (Johnson et al., 1998). The BnAO chelate HL91, another technetium complex of the amine-oxime chelate, but without a nitroimidazole substituent, also displays avid uptake in hypoxic tissue. This molecule, which carries a butyl instead of a propyl bridge between the two amine nitrogens, has been labeled with technetium-99m. Whether a mono-oxoTc(V) complex or a dioxo specie is the active complex is not known; however, electrochemical studies indicate an irreversible reduction at biologically accessible potentials (Powell et al., 1999). Initial SPECT imaging of hypoxic tumors in humans has demonstrated the utility of these complexes in identifying such lesions as "hot spots" rather than defects (Cook et al., 1998).

AGENTS FOR LUNG, LIVER, SPLEEN, BONE MARROW, AND LYMPHATICS The technetium agents for imaging the organs (liver, spleen, bone marrow) of the reticuloendithelial system (RES) are primarily aggregated albumin or colloidal materials that were developed early and that have seen little change since their introduction. Their use is based upon the highly efficient manner in which the RES is able to extract such materials from the blood stream. Approximately 80 to 90% of RES cells reside in the liver, 5 to 10% in the spleen, and the remainder in the bone marrow. Introduced by Harper as early as 1961 (Harper et al., 1964a) 99mTc-labeled sulfur colloid remains the agent of choice for this purpose. Chemically this agent consists primarily of sulfur particles although it is surmised that technetium heptasulfide may also form during the preparation (Eckelman et al., 1996). Gelatin or other agents are used to limit the growth of the particles and to constrain them to a size range of one micrometer or less. With particles greater than one micrometer, the distribution of uptake shifts towards the spleen, and for those well below a micrometer towards the bone marrow, accompanied by a longer residence time in the circulation. An increasing severity of liver disease also causes a shift of radioactivity from the liver to the spleen, bone marrow, and finally to the lungs. Alternatives to sulfur colloid that have been made available commercially include calcium phytate, a colloid that is formed in vivo (Alavi et al., 1978), tin colloids (Eckelman et al., 1996), and a preparation of microaggregated albumin of appropriate particle size (Makrigiorgos et al., 1990; Kalin et al., 1991). In each case the basic mechanism of localization remains the same. Lymph Node. The smallest colloids in routine use are those for lymphoscintigraphy, a technique that has recently grown in importance with its application to sentinel node imaging. The agent of choice is 99mTclabeled antimony trisulfide colloid (Pauli & Laub, 1937; Eckelman et al., 1996), which forms a small particle with size in the range of 100 nanometers. This agent is not available commercially at present in the US; a common alternative has been to pass a standard 99mTc-labeled sulfur colloid preparation through a 0.22micrometer filter and to administer the residual fines to the patient. These preparations probably represent a range of particle sizes and have not been well characterized. The most effective design features required for this application have not yet been elucidated. (Ege & Warbick, 1979; Kaplan et al., 1979, Wilhelm et al., 1999). Lung. The largest particles used are those of Tc-macroaggregated albumin with a nominal size of 10 to 90 micrometers of which a majority of the particles are in the range of 15 to 30 micrometers. These labeled

332

HANDBOOK OF RADIOPHARMACEUTICALS

particles become mechanically trapped in the capillary bed of the lungs with a distribution that is a function of regional blood flow. Initially the products contained particles up to 150 micrometers in length, and careful quality control was necessary. In the 1970s commercial preparations appeared making the quality control of particle size before administration a less necessary precaution. Davis (1975) has argued that the optimum agent would be microspheres, rather than aggregated protein particles, with a size range of 10 to 20 micrometers, in order that trapping be limited to the capillaries and alveoli rather than larger vessels, thereby increasing the safety factor. One commercial albumin microsphere product of a controlled size was made available in the 1970s using an ingenious double-ended vial containing a glass frit. Pertechnetate was introduced through one septum and allowed to react with the tin-coated microspheres before being withdrawn through the second septum. The labeled microspheres were then resuspended in saline essentially free of unreacted technetium. (Wagner et al., 1969). However, only particulate preparations are now available in the US. Spleen. For specific imaging of the spleen, the only technetium agent that has been used clinically is labeled autologous erythrocytes that have been slightly denatured by heat or by exposure to excess levels of tin. These damaged cells are taken up by the spleen upon re-administration. This is not nowadays a common practice. Hepatobiliary. Cholescintigraphy was an early application of radiopharmaceuticals, with an extensive list of compounds and complexes being tried in the clinic beginning as early as 1955 (Loberg et al., 1981). A complex of technetium with penicillamine was introduced in 1972 (Tubis et al., 1972), and several others, including a pyridoxyline glutamate complex (Baker et al., 1974), in 1974. The technique did not become routine, however, until the discovery of the substituted acetanilidoiminodiacetate (IDA) complexes of technetium, the first member of which was the o-dimethyl-substituted ligand (Harvey et al., 1975). This new class showed much faster hepatic transit times than previous agents, including 13ll-labeled Rose Bengal, with the result that greater contrast ratios could be achieved in a relatively short period of time. The ligands also lent themselves to a one-step "instant" kit preparation based on the original work of Eckelman with DTPA (Eckelman & Richards, 1971). Extensive evaluation of a large number of ligands of this class resulted in as many as six complexes being evaluated in humans by Loberg et al. (1981) who were also among the first to combine high performance liquid chromatography (HPLC) with radiometric detection of reaction products. Nunn used this technique to evaluate an extensive number of HIDA ligands and applied the results predictively to select the best complex (Nunn, 1983). Later under the name mebrofenin, this complex and its diisopropyl derivative (DISIDA or disofenin) became available for clinical use. There have been no further developments in designing radiopharmaceuticals for hepatic imaging with one exception, 99mTc-m-galactosyl-neoglycoalbumin (NGA), which is perhaps the first technetium agent whose distribution has been shown to reflect the presence of a specific receptor type in a tissue. This hepaticbinding protein, which occurs in mammalian hepatocytes, is specific for galactose-terminated glycoproteins. The mechanism of this binding has been very thoroughly studied over a number of years (Vera et al., 1979: Krohn et al., 1982; Stadalnik et al., 1985; Stadalnik & Vera, 2001). The concept has resulted in a closely related commercial product being developed in Japan, 99mTc-labeled human serum galactosyl albumin

TECHNETIUM RADIOPHARMACEUTICALS

?33

(99mTc-GSA), which is similar to 99mTc-NGA but has an additional three or four DTPA molecules attached to the albumin (Kubota et al., 1986; Stadalnik & Vera, 2001). These chelates allowed the development of a more classic instant kit method for routine clinical preparations. Bone. Phosphonate ligands formed the basis for therapy in such bone disorders as Paget's disease, and this targeting ability brought such ligands to the attention of Subramanian and others in the early 1970s. The diphosphonates were amenable to the recently discovered instant kit formulation, and three agents became commercially available. Of these, the technetium species formed with methylene diphosphonate (Subramanian et al., 1975) remains the most widely used. No characterization of the species formed has been made, although HPLC studies at Brookhaven National Laboratory have shown a mixture of products to be present that varies in composition with time. On this basis it is presumed that these are oligomers in solution (Steigman et al., 1977). Pinkerton et al. (1982) made an attempt to identify the most effective imaging agent represented by the different peaks isolated by HPLC, with the objective of trying to develop an improved radiopharmaceutical. Apart from this, however, no change has been made to the formulation of the clinicallyused MDP kits since they were originally introduced over twenty-five years ago. An alternative radiopharmaceutical for skeletal studies used pyrophosphate as the basis for a kit (Hosain, 1973). This radiopharmaceutical was also briefly applied to the detection of myocardial infarction (Willerson et al., 1975; Legrand et al., 1983). It is now supposed that the active ingredient in the first technetium bonescanning agent, 999mTc-polyphosphate (Subramanian & McAfee, 1971), was in fact pyrophosphate, an impurity in the samples used (Eckelman et al., 1996). The formulation of one commercial product, containing large amounts of stannous ion, inadvertently led to a means of labeling red blood cells that could be done in vivo by successive administration of the reconstituted kit followed by pertechnetate. The modern erythrocyte-labeling method (Ultra-Tag™) is an extension of this procedure, with a blood sample being incubated with a stannous tin solution, followed by oxidation of the extracellular tin with hypochlorite and the addition of pertechnetate.

RENAL AGENTS The earliest technetium renal agents were based upon different formulations containing diethylenetriaminepentaacetic acid (DTPA). The first agent employed a reaction in which ferrous ion was used to reduce the technetium. A second variant was designed to increase the rate of reaction by adding ascorbic acid, resulting in the production of an entirely different complex. The third was the kit formulation that remains the standard for imaging of glomerular filtration, the stannous chloride "instant" kit which has been alluded to in other places in this chapter because of its importance in the general design of technetium radiopharmaceutical preparations. Essentially the kit contains sufficient stannous chloride to reduce the metal and to preserve a reducing environment after complexation occurs in order to slow re-oxidation to pertechnetate; the ligand confers upon the final complex the required pharmacokinetic and targeting properties and in some cases preserves the complex by mass action. An example of this is the glucoheptonate complex, which is kinetically unstable and requires a high ligand concentration to maintain its integrity. Lowering the ligand concentration makes this material a very effective transfer or exchange labeling agent (Carroll et al., 1985).

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HANDBOOK OF RADIOPHARMACEUTICALS

The complexes formed with glucoheptonate, dimercaptosuccinic acid (DMSA) and the iron-ascorbate reduction product with DTPA undergo a mixture of glomerular filtration, tubular secretion and retention in the kidneys. These so-called mixed-function agents (Eckelman et al., 1996) are used generally to examine the patency of the renal cortex. It has been noted that the presence of stannous ion tends to increase this effect (Ikeda et al., 1977; Fritzberg et al., 1978; Steigman et al., 1979) though without any specific mechanism being identified. -1

-2

-1

O'

HOTc(IV>DTPA

Tc(V)-DTPA

'CH2OH

Tc( V>Glucoheptonate

Proposed structures for Tc-DTPA and Tc-Glucoheptonate These tracer-level complexes have not been positively identified chemically, although the crystal structure of one of the isomeric complexes formed by meso-DMSA (methyl ester) has been established (Bandoli et al., 1984). The stereochemistry of this system and of other technetium species has been extensively discussed in the literature (Nowotnik & Jurisson, 1992; Bandoli et al., 2001) The form of the glucoheptonate complex with technetium-99m has been deduced from various pieces of evidence, including a comparison of the complexes formed at tracer and carrier-added concentrations with technetium-99, as being an oxotechnetium(V) bisligand anion (de Kieviet, 1981). No X-ray-structural data was collected but the same complex was apparently detected at both macro and tracer concentrations. This is somewhat at variance with other electrochemical and polarographic results that indicate a possible mix of species in solution (Russell & Speiser, 1980). There is, thus, no certainty as to the nature of the species being employed for imaging. The more recent 99mTc-MAG3, on the other hand, has been fully characterized. This molecule stemmed from efforts to design suitable tetradentate chelates to sequester the metal that could 'be modified with other moieties which might guide the complex to a variety of target tissues. The first of these, TcO(ema) or TcDADS, showed rapid clearance through the kidneys after administration but also a small percentage in the intestines (Davison et al., 1981). Attempts to improve on this behavior led to the ligand CO2DADS which gave two complexes with the metal that were separable by chromatography and were shown by mass spectrometry to be identical (Costello et al., 1983). Fritzberg also showed that the complex was primarily cleared through the kidneys by secretion, raising the prospect of a technetium replacement for ortho-iodohippuric acid (Fritzberg et al., 1982). Other related ligands in this series were also investigated (Nowotnik & Jurisson, 1992) but eventually the nonchiral ligand

TECHNETIUM RADIOPHARMACEUTICALS

335

[N-[N-[N-(mercaptoacetyl)-glycyl]-glycyl]glycine] was developed (Fritzberg et al., 1986). This compound also forms a mixture of enantiomers, each of which has been studied individually in primates and in man and all of which show essentially the same biologic behavior (Verbruggen et al., 1989). Tc-MAGj remains the standard radiopharmaeeutical for renal function imaging today.

RECEPTOR-SPECIFIC AGENTS

-I -2

Tc-DADS

Tc-COjDADS

Tc-MAG3

Localization in target organs of the earlier technetium complexes, such as 99mTc-MDP, 99mTc-HIDA, 99mTcDTPA, and

99m

Tc-HMPAO, was primarily based on properties such as the size, charge, lipophilicity, or

hydrophilicity, and other gross targeting/localizing characteristics imparted by the ligand to the overall complex. As the role of in vivo characteristics such as blood clearance, protein binding, in vivo stability, and the physiologic mechanism necessary for localization became apparent in the design of radiopharmaceuticals, complexes such as [99mTc-ECD], [99mTc(I)(MIBI)6]+ and [99mTc-MAG3]-2 were synthesized. As such agents were studied further both in the laboratory and the clinic, it became apparent through mechanistic studies that specific interactions of these technetium complexes with particular enzymes and specific cellular proteins also occur, e.g., the hydrolysis of the ester in 99mTc-ECD by esterases in the brain of primates and humans (Walovitch et al., 1989; Walovitch et al., 1991), the recognition that certain tumors accumulate [99mTc(IXMIBI)6]+ (Delmon-Moingeon et al., 1990) and, more important, that this lipophilic cation can also act as a substrate for P-glycoprotein, one of a class of proteins responsible for imparting mutlidrug resistance to tumors (Piwnica-Worms et al., 1993). Although not original design criteria, these observations were the first indication that technetium (i.e. metal)-based radiopharmaceuticals could be designed to image specific enzyme or protein interactions. Until recently, receptor imaging in nuclear medicine was almost exclusively performed with ligands radiolabeled with isotopes such as iodine-123, fluorine-18 and carbon-11. The high economic costs and isotope unavailability for routinely producing receptor-specific imaging agents with these isotopes provided the impetus for similar agents labeled with the more widely available technetium-99m. However, the labeling with technetium inherently necessitates the incorporation of a chelate or coordinating atoms within, or appended to, the molecular framework of the receptor ligand for complexing technetium. Although this presents a formidable problem, efforts by several groups have shown that the challenge is not insurmountable.

336

HANDBOOK OF RADIOPHARMACEUTICALS

In the late 1980s an increasing emphasis on designing technetium radiopharmaceuticals that have specific interaction with cellular proteins and/or receptors began to emerge. Conjugates of technetium complexes with monoclonal antibodies and their fragments that target cellular antigens have been explored, and various strategies based on both direct and bifunctional chelate conjugates have been developed (Liu & Edwards, 1999). While most monoclonal antibodies display high affinity for their antigen in vitro, their inherent size and inadequate pharmacokinetics in vivo have hindered their widespread use in human studies. Nevertheless, various 99mTe-labeled antibody fragments have been used clinically to image cancers and melanoma (Serafini, 1993). Although not yet used clinically, interesting results have recently been reported with the use of a 99mTc-labeled, small 36 kDa endogenous protein, annexin V. This protein binds to phosphoserine headgroups in phosphatidyl serine (PS) with an affinity of about 10-9 M. While PS is a major innermembrane constituent of most cells, the initiation of apoptosis (which may be a result of various other factors) results in the inactivation of membrane pumps such as translocase and floppase that actively control the localization of PS, and activation of an enzyme scramblase, which equilibrates the inner and outer membrane lipids including PS on the cell surface (Tait, 1996). Annexin V has been labeled with technetium99m by hydrazino nicotinamide (HYNIC) conjugation and, after intravenous injection, has been shown to clear rapidly from circulation with a half-life of about five minutes. Blankenberg et al. (1998) have used 99m Tc-annexin V to detect apoptosis in vivo in animal models of transplant rejection, to visualize apoptotic cells in Fas-mediated apoptosis, and to assess chemotherapy of xenographs. The potential for using 99mTclabeled annexin V to visualize apoptosis in a wide array of pathologies is clearly unique and worth further investigation (Blankenberg et al., 2000). The recognition of the importance of the in vivo pharmacokinetic parameters necessary for drug delivery to a particular target and a greater understanding of the basic chemistry of technetium have led to the development of a number of receptor-specific technetium complexes (Hom & Katzenellenbogen, 1997a; Jurisson & Lydon, 1999). The general design criteria applied in the development of these agents are largely dictated by the location of the target protein in the organ/body, whether vascular, peripheral, or within the central nervous system, as well as whether the receptors are expressed intra- or extracellularly. This in turn determines the nature and size of the 99mTc-labeled agent that can be delivered efficiently to the required location. Obviously, targets in the vasculature such as thrombi and atherosclerotic plaque are more accessible than neuroreceptors and transporter proteins in the brain and extracellular membrane receptors are more accessible than intracellular protein targets. In addition, other parameters that ultimately determine the image quality include the receptor density and the affinity of the final complex versus background or nonspecific binding. Receptor-specific technetium complexes can be broadly divided into two classes, namely agents based on small peptides and those based on small organic molecules. In the former case, technetium is attached via a chelate incorporated within/appended to the peptide in a region that does not interfere with the affinity of the peptide for the receptor. In general, these peptides are small (5 to 12 amino acids) and may be cyclic or linear. In the latter case the molecules are even smaller in size and are generally organic ligands in which the

TliCHNETlUM RADIOPHARMACEUT1CALS

337

technetium complex is appended to a sterically tolerant region of the molecule or integrated within the whole molecule to mimic the three-dimensional structure of the receptor ligand. The receptor-specific labeled peptides that have been developed so far, owing to their size, are not surprisingly targeted to receptor proteins that are easily accessible following intravenous administration, e.g. those on deep vein thrombosis (DVT) and pulmonary emboli in the vasculature, or over-expressed on the cell-surface of tumors accessible to blood. Receptor-specific small-molecule complexes, on the other hand, mainly include those developed for neuroreceptors, transporter proteins or steroid receptors as targets.

TUMOR AGENTS The first radiolabeled peptide to enter clinical use for tumor imaging is 111in-DTPA-octreotide (OctreoScan™), This 111in-labeled somatostatin analogue is a disulfide-bridged cyclic 8-amino-acid analogue that displays somatostatin-receptor affinity with a Ki = 1.2 nM. Despite the poor imaging characteristics of indium-111, the agent is routinely used for imaging tumors of neuroendocrine origin and pulmonary neoplasia (Krenning et al., 1994). Based on this structure, synthetic analogues containing the cyclic -Tyr-(DTrp)-Lys-Val- receptor-binding sequence but with a covalent nonreducing cyclic amide instead of the disulfide group were developed by Lister-James et al. (Lister-James et al, 1996a; Lister-James & Dean, 1999) and labeled with technetium-99m. Among these peptides, P829 (Kd = 1.5-2.5 nM) has shown considerable promise in imaging primary and metastatic nonsmall-cell lung cancer (Leimer et al, 1998), Hodgkin's and nonHodgkin's lymphoma (Line et al., 1998; Virgolini et al., 1998; Lister-James & Dean, 1999), and breast cancers. The agent was recently approved by the FDA for tumor imaging.

[111lnDTPA]-D-Phe— Cy Cys

^ D-Trp

D-Tr Trp

/

/ (NMe)Phe- Tyr

/

Thr

[111 In-DTPA]-Octerotide

P829

In an analogous fashion to somatostatin, vasoactive intestinal peptide receptors (VIP) are also over-expressed in a variety of endocrine tumors, adenocarcinomas, lymphomas and in melanoma. (McAfee & Neumann, 1996). An analogue of VIP, P1666, containing a technetium-chelating tripeptide sequence [desMet17, ~S(CH2CO-Gly-Cys-Lys-amide)-Hcy17]VIP, has been explored for imaging colorectal cancer (Lister-James et al., 1998).

338

HANDBOOK OF RADIOPHARMACEUTICALS

Cyclic99mTca-MSH analogue

Another peptide-hormone receptor expressed on murine and human melanoma tumors is the a-melanocytestimulating hormone (a-MSH) receptor, which is responsible for regulating melanin production. The a-MSH peptide, a linear tridecapeptide (Ac-Ser'-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Glylo-Lys11-ProI2-Val13NH2) has also recently been modified by substitution of D-Phe7 and cyclized via metal (Tc or Re) coordination of cysteine residues introduced at the N- and C- termini to produce enzymatically stable technetium and rhenium analogues that display significant tumor localization in vivo (Giblin et al., 1997; Giblin et al., 1998; Chen et al., 2000).

The integrin receptors, particularly the OvP3 (also known as the vitronectin receptor), bind to proteins and peptides containing the RGD (-Arg-Gly-Asp-) sequence. Many vitronectin antagonists inhibit tumor-induced angiogenesis and tumor growth (Stromblad & Cheresh, 1996). A synthetic linear peptide oP2 (Arg-Gly-AspSer-Cys-Arg-Gly-Asp-Ser-Tyr) containing two RGD sequences has also been recently labeled with technetium-99m. While the mode of technetium coordination has not been reported, studies have demonstrated rapid clearance via the renal system and successful imaging of lymph node metastases (Vamer & Cheresh, 1996). Similarly, RP593, a dimeric vitronectin receptor antagonist containing two con formation ally constrained RGD sequences, was designed and labeled with technetium-99m/technetium-99 (Liu et al., 2001). In vitro nectin-receptor assays were performed in vitro, a high affinity (IC50 = 1.7 ± 0.5 nM) was observed, and preliminary studies in animal tumor models indicate rapid blood clearance with tumor uptake of 5.5% injected dose/gram observed at one hour after injection (Barrett et al., 2000; Janssen et al., 2000).

TECHNETIUM RADIOPHARMACEUTICALS

339

AGENTS FOR INFECTION AND INFLAMMATION Imaging sites of inflammation and infection was initially performed with [67Ga]gallium citrate or by labeling autologous white blood cells (WBC) in vitro using 111In(oxine)3 (Seabold et al., 1997a and b). The large absorbed radiation dose and potential radiation damage to long circulating lymphocytes coupled with the poor imaging characteristics of the isotope led to the development of procedures to label WBC with 99mTcHMPAO (Datz et al., 1997). Both procedures are still in use; however, they are cumbersome and pose potential risks of transmission of blood-borne diseases such as HTV to both patients and personnel. The use of 99mTc-labeled small chemotactic peptides for targeting sites of infection/inflammation has also been explored in animal models. These agonists, however, are quite potent and induce a transient yet severe neutropenic response. More recent advances in developing 99mTc-labeled peptides include the development of tuftsin, a cationic tetrapeptide (Tyr-Lys-Pro-Arg) derived from leukokinin (Caveliers et al., 2001). The tetrapeptide was modified at the N-terminus with the additional tetrapeptide sequence X-Ser-Cys-Gly (X = dimethylGly [RP128]) to provide a N3S coordinating environment (X-Ser-Cys-) for chelating technetium coupled with a glycine spacer to the tuftsin antagonist, -Tyr-Lys-Pro-Pro-Arg. Preliminary human studies with this 99mre-labeled agent indicate rapid renal clearance and good target to background ratios within three to four hours in patients with Crohn's disease and rheumatoid arthritis (Iles et al., 1998). HO

., 99

"Tc-RP128

P483H

Similarly, another 23-amino-acid cationic peptide P483H, derived from the heparin-binding region of platelet factor 4 (PF–4), a 29 kDa heterotetrameric protein, has been modified to include a -Cys-Gly-Cys-(N2S2) tripeptide-chelating sequence within the peptide (Moyer et al., 1996). In combination with heparin this 99mTclabeled peptide has improved significantly the 99mTc-labeling of WBC, particularly monocytes, and permits imaging of sites of infection in a rabbit infection model. Among the other 99mTc-labeled agents that have been evaluated for imaging infection, a labeled antibiotic (ciprofloxacin), has recently been reported as a radioligand for visualization of bacterial infection (Britton et al., 1997). While the technetium labeling yield is only 40% and the exact mode of technetium coordination has not been determined, it has been evaluated in humans as a candidate for imaging bacterial infection (De Winter et al., 2001).

THROMBUS AGENTS In a rapidly growing thrombus, activated platelets express GP llb/TIIa receptors that recognize peptides and proteins such as fibrinogen containing a tripeptide RGD (-Arg-Gly-Asp-) sequence (Ojima et al., 1995). A number of 99rnTc-labeled antithrombotic GP Ilb/IIIa receptor antagonists have been developed and evaluated for the noninvasive imaging of thromboembolism. DMP 757, an RGD-containing, conformationally

340

HANDBOOK OF RADIOPHARMACEUTICALS

constrained, cyclic GP Ila/IIIb antagonist which displays high affinity (6 nM) binding to activated platelets, has been modified to append chelates such as N2S2 (RP419) and HYNIC/tricine/TPPTS (DMP444) which permit technetium labeling (Barrett et al., 1996; Barrett et al., 1997; Edwards et al., 1997).

DMP7S7

Both 99mTc-labeled agents have been evaluated in a canine AV shunt model and display rapid uptake in thrombi which were clearly visualized within 15 to 30 minutes after injection (Barrett et al., 1997; Liu et al., 1997). Although DMP 444 displays two isomeric complexes upon labeling, both isomers have a 12 nM affinity against fibrinogen binding to activated canine platelets (Liu & Edwards, 1999). Phase II clinical trials indicate that DMP 444 could detect deep vein thrombosis (DVT) within 60 minutes with an 82% accuracy, 93% sensitivity, and 75% specificity (Line et al., 1996).

P280

v

...NH Gly'

G

6

^P

(Amp=4-am«Jino-pheny1analine)

.Gly, D-Tyr P748

TECHNETIUM RADIOPHARMACEUTICALS

341

The dimeric GPIIa/IIIb receptor antagonists P280 and P748 which contain 3-aminopropyl-cysteine and 4amidino-phenylalanine, respectively, instead of arginine in the RGD sequence have also been reported as high affinity ligands for imaging DVT and pulmonary embolism (Lister-James et al., 1996b; Pearson et al., 1996). Upon complexation with technetium-99m, the dimeric unit in P280 is converted to a monomeric unit (Lister-James & Dean, 1999) which has an IC50 of 6.8 nM against fibrinogen binding to GPIIa/IIib, and an IC50 of 79 nM for platelet aggregation inhibition. Similarly, P748 shows an IC50

D-Tyr Ap

Gly—Asp

"",

^

'„

, ^NH-Cys(Acm)-Gly-Cys(Acm>-

O 99m

Tc-P280

H2N

O

of 28 nM for platelet aggregation inhibition (Lister-James et al., 1996b). 99mTc-P280 has recently been approved for DVT imaging and is commercially available as AcuTec™ from Nycomed-Amersham. Linear RGD-containing peptides derived from the CDR region of a PAC 1.1 monoclonal antibody such as CYT379 (Ac-Ser-Tyr-Gly-Arg-Gly-Asp-Val-Arg-Gly-Asp-F-Lys-Cys-T-Cys-Cys-Ala-NH2) include two RGD sequences, and these have also been labeled with technetium-99m (Rodwell et al., 1991; Knight et al., 1994).

AGENTS FOR NEURORECEPTORS/TRANSPORTERS AND AMYLOID PLAQUE Noninvasive neuroreceptor imaging could greatly aid in the understanding, diagnosis, and monitoring of various neurologic diseases. Biochemical studies on neuroreceptors and recent advances in structural and computational methods in designing drugs and ligands, coupled with tissue-distribution studies of these drugs labeled with beta- and positron-emitting isotopes, have provided a reliable foundation and facilitated the development of criteria upon which investigations of new technetium-based neuroreceptor-specific radiopharmaceuticals may be based. An important consideration in designing such radiopharmaeeuticals is that, unlike the accessibility of receptor proteins in the vasculature and in peripheral tissue, accessibility to the neuroreceptors is restricted by the blood-brain barrier. Thus, targeting these receptors with 99mTccomplexes, which inherently require a chelate, also demands that the complexes be small, neutral, lipophilic and stable in vivo, as well as possess high affinity for the targeted receptor with minimal nonspecific binding to other proteins and receptors. Designing 99mTc-complexes within these parameters and meeting the threedimensional structural requirements necessary for high affinity binding to the receptor proteins have posed a significant challenge. Considerable efforts have been made within the last decade and a number of 99mTc-complexes possessing moderate to high affinity for neuroreceptors have been reported (Hom & Katzenellenbogen, 1997a; Jurisson & Lydon, 1999). The two most promising of such agents, both targeting the dopamine transporter (DAT)

342

HANDBOOK OF RADIOPHARMACEUTICALS

protein and both being tropane derivatives, have shown receptor-specific localization in primate and human brains (Madras et al., 1996; Kung et al, 1997; Bonab et al., 2000; Bonab et al., 2001). O —N.

—N

ocr'cpo X-I, F, B-CFT

Cocaine

rs

TROTEC

X

"Tc-TRODAT

99m

Tc-Technepine X- OMe (O861T)

"Tc-Technepine X= Et (O1505T)

Dopamine transporter protein (DAT): Dopamine transporter, a key neurotransporter located presynaptically in high density at dopaminergic nerve terminals in the basal ganglia, plays a vital role in regulating the endogenous dopamine concentration in the synaptic cleft. Based on the structural derivatives of tropanes which display high affinity for DAT (P-CIT, P-CFT, FPCIT, altropane) a number of 99mTc-labeled DAT radiopharmaceuticals have been synthesized and shown to display moderate to high affinity for the transporter (Madras et al., 1996; Meegalla et at., 1997; Hoepping et al., 1998; Meltzer et at., 2000). Although most of these complexes have low brain uptake, 99mTc-TRODAT and 99mTc-technepine have sufficient brain uptake and localization to permit in vivo visualization of DAT distribution in the primate and human brain (Kung et al, 1996; Bonab et al., 2001). 99m

Tc-TRODAT is a tropane derivative in which the 2p-methyl ester is substituted with a methylene-1inked tetradentate bisaminoethanethiol chelate in the 2|3-position of the tropane. Complexation with technetium or

rhenium results in neutral lipophilic diasteriomeric (R and 5) complexes, both with the receptor-binding Nmethyl 3p-(4-chlorophenyl)tropane moeity in a syn configuration with respect to the Tc=O group. The R isomer displays a slightly higher lipophilicity (log P = 2.48) and a lower binding affinity (K, = 13.87 ± 1.7 nM) relative to the 5 isomer (log P = 2.35) which has a Ki of 8.42 ± 0.67 nM (Meegalla et al., 1998). 99mTcTRODAT has affinity for the serotonin transporter (SERT) as well, with a Ki of 360 ± 44 nM. The selectivity of 99mTc-TRODAT for DAT versus SERT is thus approximately 25 for the mixture of R and S, which means that a significant concentration of SERT is visualized with this agent. This aspect of 99mTc-TRODAT affinity for SERT has been used in imaging the SERT in the midbrain region of nonhuman primates by specifically blocking the DAT (Dresel et al., 1999). 99m

Tc-technepine, also a tropane analogue, is the first99mTc-tropanederivative to display distinct localization

of DAT in nonhuman primates (Madras et al., 19%). A tetradentate amine-amide-dithiol is appended to the amine nitrogen of 2p-methylester-3p(4-fluorophenyl)tropane via a three-carbon linker (Meltzer et al., 1997; Meltzer et al., 2000). The technetium and rhenium complexes form only the syn geometric isomers with R

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and 5 configuration. The R and S diasteriomers possess slightly differing affinity for DAT (1R, 12S: Ki = 7.32 ± 1.33 nM, 1R,12R: Kt - 4.04 ± 0.98 nM) and considerably different affinity for SERT (Ki - 66.9 ± 3.0 nM and 299 ± 23 nM, respectively), thus resulting in DAT/SERT selectivity of 9 and 74 for the two isomers. While the first generation technepine permitted distinct visualization of DAT in the striatum of nonhurnan primates, the absolute uptake in the brain was low. Further refinement of the structure has subsequently resulted in the second generation technepine which contains not only a tropane in the boat configuration (rather then a chair configuration as in the other tropanes) but also an ethyl ketone in the 2(3-position (Meltzer et al., 2000). These modifications have improved brain uptake significantly in both human and nonhuman primates, permitting adequate imaging. They have also increased the selectivity for DAT (IC50 = 2.0 nM) versus SERT (IC50 = 497 nM), an order of magnitude higher than TRODAT (249 versus 25), thus providing a means for noninvasively imaging DAT in neurologic disorders without confounding SERT uptake (Bonab et al., 2000; Bonab et al., 2001). Serotonin 5HT/a and 5HT2a receptors: Serotonin receptors are another class of neuroreceptor for which technetium-99m based receptor-specific complexes are being designed. Alterations of serotonin receptors are implicated in a variety of anxiety disorders, depression, schizophrenia and Alzheimer's disease. While many subtypes of this receptor are known, the 5-HT1A and 5-HT2A have received the most attention with respect to developing 99mTc-labeled receptor-specific complexes. Most of the complexes investigated for the serotonin 5-HT1A receptor are analogues of WAY 000635 or p-MPPl, both of which are high affinity antagonists at the 5-HT1A receptor and possess a l-(2-methoxyphenyl)piperazine (MPP) as the receptor-binding moiety.

A number of variations with respect to the linkage and type of chelate have been investigated. Lever and Baidoo have reported several TcvO complexes of MPP linked to a tetramethyl or hexamethyl diamine-dithiol N2S2 chelate via alkyl amides of varying length (Baidoo et al., 1995; Baidoo et al., 1997). Although the butylamine-linked hexamethyl N2S2 complex (1) displays an in vitro affinity with Ki of 10.4 nM for the 5HT1A receptor, in vivo brain uptake is low (Baidoo et al., 1995). Similar brain uptake values have been obtained by Kung et al. (1995) where the reported complexes contain both the 2-pyridyl substituent (as in WAY100635) and the aromatic benzamide (as in p-MPPI) linked to a less lipophilic amine-amide N2S2 TcO complex (2) and possess a Ki of 25 nM (Kung et al., 1995). Complexes designed to integrate the aromatic group within the chelate display lower affinity (Ki = 146 nM) (Ova et al., 1996).

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Mahmood et al. (1999) have reported several MPP-linked TcvO complexes with both amine-amide and diamine N2S2 chelates, where the removal of the aromatic 2-pyridyl group (as in WAY 10063) has been explored. While MPP linked through a propyl amide (3) gave a moderate in vitro affinity (IC50 = 30 nM), further modification of the structure by converting the alkylamide bond to a simple alkyl (propyl) provides both amine-amide (4) or diamine N2S2 complexes (5) with high in vitro affinity (IC50 = 3.6 and 4.4 nM, respectively) for the 5-HT!A receptor (Mahmood et al., 1999). Despite the high in vitro affinity for the 5HT]A receptor, low in vivo brain uptake in rats was observed, precluding further evaluation in primates. Technetium complexes have been designed by Johannsen et al. (19%) to target the serotonin 5-HT2A receptor. "3+1" complexes containing TcvO (6) have been explored using structural elements of ketanserin, a known 5-HT2A ligand. Again, despite high in vitro affinity (7 nM) for the receptor, the in vivo brain uptake is low.

Ketanserin

Amyloid Plaque: Technetium complexes that target other proteins involved in neurologic disorders, such as amyloid plaque, are also under investigation. While it is not clear whether amyloid plaques are the result or the cause of neuronal degeneration in Alzheimer's disease, staining by aromatic azo dyes such as congo red and chrysamine G displays extensive amyloid deposits in postmortem brain tissues from Alzheimer's patients (Ashburn et al., 1996). This has led investigators to evaluate various modes of incorporating the basic dye structure with technetium complexes (Han et al., 1996). An integrated approach has been described by Lansbury and Kosik (2000) where a central bipyridyl group capable of bidentate coordination to technetium is introduced in lieu of the biphenyl group in the azo dyes. Mixed-ligand tetrakis(t-butylisonitrile)Tc(I) complexes have been formed with these bipyridyl dyes using the hexakis(t-butylisonitrile)Tc(I) complex that displays moderate affinity to ^-amyloid fibrils (Kd = 160–700 nM). The charges on the complexes, however, render them unsuitable for further in vivo studies.

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Alternate approaches using pendent N2S2 chelates have also been reported for these azo dyes as well as their uncharged ester derivatives (Dezutter et al., 1999; Zhen et al., 1999). Although in vitro staining and autoradiography indicate labeling of vascular and amyloid plaques in postmortem brain tissue, in vivo studies in mice show low brain uptake even for the apparently neutral ester derivatives (Dezutter et al., 1999). Sigma Receptors: Originally thought to be a subclass of opiate receptors, sigma receptors are nondopaminergic, nonopiate membrane receptors that possess high affinity for haloperidol and various other neuroleptics (Walker et al., 1990). Two subtypes, termed o—1 and a-2, have now been identified, with the O)~benzomorphans. (+)~[3H]-pentazocine selectively labels the o~T sites. The a-2 sites, on the other hand, are identified with a nonselective a-l/o-2 ligand, [3H]DTG in the presence of dextrallophan, which masks binding of the o-l sites. Other distinguishing characteristics include the apparent molecular weight differences, the sensitivity to guanine nucleotides, and the allosteric modulation with rozipine and phenyltoin (Walker et al,, 1990). While the exact functional role of these receptors is not completely understood, a number of ligands for this G-protein-coupled receptor have been labeled with fluorine-18 and carbon-11 for mapping their in vivo brain distribution (de Costa et al., 1992; Ishiwata et al., 1998). The expression of these receptors on a number of human and murine tumors (Vilner et al., 1995) has also led to evaluation of these PET radioligands in cancer imaging (Maffioli et al., 1994; John et al., 1999). This in turn has prompted the investigation of technetium complexes that could bind to such sigma receptors. In this regard, based on earlier structure-activity studies

of aminoalkyl pyrrolidine and piperidine ligands, John et al. (1997) designed a Tc(v)ON2S2 complex (99mTcBAT-EN6) with the necessary 2-(N'-methyl)aminoethylpiperidine receptor-recognition elements as an Nsubstituent on the complex. The technetium complex forms both syn and and isomers in a 55:45 ratio and displays high specific binding to the sigma receptors expressed on human-ductal-breast-carcinoma (T47D)

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cell membranes. Dose-dependent inhibition has also been observed with known sigma ligands; however, neither binding of individual isomers nor independent o-l and a-2 affinities have been measured. A combined noncompetitive saturation binding at both the a-1 and a-2 receptor with Kd of 43.5 ± 14.7 nM has been observed and a competitive Ki of 42.7 ± 8.5 against [3H]DTG. Although no in vivo tumor studies have been reported with this complex, in vivo inhibition of receptor binding in the kidney, lung, and liver, organs known to express both a-1 and a-2 receptors, has been observed when BD1008, a sigma ligand. is coadministered with the technetium complex.

Granatane

Recently another technetium complex (7) with selective a-2 affinity has been reported by Mach et al. (2001). These complexes have been designed on the basis of earlier work that demonstrated that tropane and granatane derivatives display significant selectivity and affinity for a-2 receptors. As the bridgehead nitrogen of the granatane has been shown to accommodate steric bulk without loss of affinity, an amineamide-dithiol N2S2 chelate has been appended to the granatane derivative via a propyl linker. In vitro a-receptor affinities of Ki = 2723 nM and 22 nM for the a-1 and a-2 receptors, respectively, have been reported. Selective in vivo accumulation in tissues where o-receptors are abundant has also been observed (Choi et al., 2001). Steroid receptors: Technetium complexes that target the steroid receptors have been an area of active research as estrogen, progesterone, and androgen receptors are known to be over-expressed in breast, ovarian, and prostate cancers. Katzenellenbogen and coworkers have done extensive research with steroidreceptor ligands, and they have evaluated various strategies in designing technetium-labeled complexes targeting this receptor.

Their first reported technetium complexes were progestin conjugates of N2S2 chelates based on RU486 (DiZio et al., 1991; DiZio et al., 1992). Although the ami isomer of the tetramethyl N2S2 complex (8) appended via a benzyl link to the 11P-position of the steroid had an estimated affinity of 2 nM at the progestin receptor, higher then progesterone itself, excessive lipophilicity (log P = 6.5) of the complex induced high nonspecific binding and excessive uptake in the liver and adipose tissues. The use of less

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lipophilic amine-amide-dithiol N2S2 chelates reduced the lipophilicity of the complex (9) and maintained moderate binding to the progestin receptor. However, the target-tissue uptake relative to the liver and other organs was not adequate (O'Neil et al, 1994). Considering the intracellular location of the target receptor and the membrane lipid bilayer of the cell that the technetium complex must traverse necessitates a design that balances the size and lipophilicity of the complex with the receptor-binding affinity.

H ~ " ' H 5a-dihydrotestosterone (DHT) Bis-bidentate DHT mimic

Estradiol

Estradiol C7 - C8 mimic

Estradiol CI3 - C !4 mimic

Attempts to reduce the molecular mass and lipophilicity have led to the design of integrated complexes with the oxometal group integrated within the B,C or C,D rings of 5 a-dihydrotestosterone or estradiol, respectively. Both bidentate mixed-ligand Tc(V)O complexes and tetradentate Tc(V)O complexes which project the functional groups around the complex have been investigated as molecular mimics for these steroids. However, these attempts have also met with limited success, due to low stability and/or low affinity of these complexes for the respective steroid receptors (Horn & Katzenellenbogen, 1997a and b).

MULTIDRUG RESISTANCE Multidrug resistance (MDR) is a phenomenon in which cancer cells become resistant to a structurally diverse array of chemotherapeutic drugs with differing modes of action. Different mechanisms have been characterized in MDR, and it is, therefore, considered a multifactorial phenomenon (Germann, 1996; Lehnert, 1996). One of the most studied of these mechanisms is the expression or over-expression of an ATP-dependent P-glycoprotein (Pgp), which is encoded by the MDR1 gene located on human chromosome 7 (7q21.1). Pgp is a 170-180 kDa transmembrane protein consisting of two similar halves, each of six transmembrane domains with two intracellular ATP-binding sites (Hendrikse et al., 1999). A second mechanism in MDR, quite distinct from the expression of Pgp, is the expression of a multidrug-resistanceassociated protein (MRP1), encoded by the MRP] gene located on chromosome 6 (16pl3.1). MRP1, a 190 kDa protein, is also a member of the ATP-binding cassette of transport proteins and functions as a glutathione S-conjugate efflux pump, transporting drugs conjugated or co-transported with glutathione outside the cell. While both proteins function as ATP-dependent drug efflux pumps, their mechanism of action and distribution within various tissues and tumors differs (Hendrikse et al., 1999).

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Since resistance of various cancers to chemotherapy is a major cause of treatment failure, determining the resistance status of a tumor has considerable prognostic value. While various in vitro detection assays are available for estimating MDR status in clinical tumor biopsies and in various parental and resistant tumor cell lines (Broxterman et al., 19%), functional measurement of the MDR status in vivo via SPECT or PET imaging would represent a major advantage over conventional in vitro techniques (Hendrikse et al., 1999; Sharma & Piwnica-Worms, 1999). The observation that the myocardial perfusion agent [99mTc(I)(MIBI)6]+ is a transport substrate for Pgp and MRP1 (Piwnica-Worms et al., 1993; Sharma & Piwnica-Worms, 1999) has prompted several investigations to evaluate the potential use of this agent in the functional monitoring of the MDR status. Studies in both animal tumor models (Piwnica-Worms et al., 1993; Ballinger et al., 19%; Barbarics et al, 1998a and b) and in prospective human trials (Kostakoglu et al., 1997; Luker et al., 1997; Kostakoglu et al., 1998) indicate not only a significant decrease in 99mTc(MIBI)6+ uptake in resistant tumors but also an increased washout rate from tumors expressing Pgp. The use of MDR reversal agents such as PSC833, a nonimmunosuppressive cyclosporin analogue, in modulating the net uptake and clearance rates of 99mTc(MIBI)6+ in Pgp-expressing tissues including resistant tumors has also been studied, demonstrating the effects of these reversal agents on tumors and other Pgp-expressing tissues (Luker et al., 1997; Barbarics et al., 1998a and b). Other lipophilic cationic complexes of technetium such as99mTc-tetrofosmin (Ballinger et al., 19%) and the 99m

Tc-Q complexes have also been shown to be substrates for Pgp in both in vitro and in vivo animal tumor

models. Efforts to improve the sensitivity of the mixed-ligand Q complexes by varying the planar tetradentate Schiff base and axial phosphine ligands have identified three variants of the99mTc-Qcomplexes, namely

99m

Tc-Q57,

99m

Tc-Q58 and

99m

Tc-Q63, that display low nonspecific binding, a relatively high

differential uptake between drug-sensitive and drug-resistant tumors, and a high enhancement of uptake in combination with cyclosporin A, an MDR modulator (Crankshaw et al., 1998).

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While the clinical methodology for measuring the MDR status of various tumors in humans is still evolving, the measurements are not direct but rather relative and entail measurement of the net tumor accumulation of 99m

Tc(MIBI)6+ (before and after chemotherapy); measurement of the relative washout rates of 99mTc(MIBI)6+

from Pgp-expressing tissue; and relative modulation of

99m

Tc(MIBI)6+ uptake in Pgp-expressing tissues

before and after treatment with reversal agents such as PSC833, cyclosporin A, or other MDR-reversal agents. It has been suggested that a careful analysis of this data can provide a measure of the MDR status of the tumors (Kostakoglu et al., 1997; Luker et al., 1997; Ciarmiello et al., 1998; Kostakoglu et al., 1998; Del Vecchio et al,, 2000). CONCLUSION The discussion above is not intended as a comprehensive survey of the literature but is meant to provide a general overview of the field, from the time when little was known about technetium chemistry and most technetium radiopharmaceuticals were uncharacterized to the present day when technetium chemistry is known in considerable detail and radiopharmaceuticals are routinely characterized species. The enormous amount of work by many individuals and research groups has contributed greatly to insights into the influence of the various chemical and structural variations displayed by these complexes and the effect these have on their physiochemical properties and biologic behavior. As the field of technetium radiopharmaceuticals has progressed, we have gone from direct labeling of proteins and monoclonal antibodies to more specifically designed peptides labeled with technetium for imaging various pathologies; from technetium complexes that measure perfusion and blood flow to complexes designed for measuring specific receptor density and/or expression. Further developments in elucidating new chemistries and coordination environments (Alberto et al., 1998; Schibli et al., 2000) coupled with devising new methods for labeling biologically relevant molecules (Pollak et al., 1999; Dunn-Dufault et al., 2000) are continually being added to extend the repertoire in technetium radiopharmaceutical chemistry.

ACKNOWLEDGEMENTS The authors wish to thank the untiring efforts of our editor, Rebekah A. Taube, MA , in the finalizing of this manuscript. Partial support for this work was derived from USPHS Grant RO1 CA34970.

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Verbruggen A, Bormans G, Cleynhens B, Hoogmartens M, Vandecruys A and De Roo M (1989) Separation of the enantiomers of 99mTc-MAG3 and their renal excretion in baboons and a volunteer. Nuklearmedizin, Suppl. 25, 436–439. Vilner BJ, John CS and Bowen WD (1995) Sigma-1 and sigma-2 receptors are expressed in a wide variety of human and rodent tumor cell lines. Cancer Res., 55, 408–413. Virgolini I, Leimer M, Handmaker H, Lastoria S, Bischof C, Muto P, Pangerl T, Gludovacz D, PeckRadosavljevic M, Lister-James J, Hamilton G, Kaserer K, Valent P and Dean R (1998) Somatostatin receptor subtype specificity and in vivo binding of a novel tumor tracer, 99mTc-P829. Cancer Res., 58, 1850–1859. Wagner HN Jr, Hosain F and Rhodes BA (1969) Recently developed radiopharmaceuticals: ytterbium-169 DTPA and technetium-99m microspheres. Radial. Clin. North Am., 7, 233–241. Walker JM, Bowen WD, Walker FO, Matsumoto RR, De Costa B and Rice KC (1990) Sigma receptors: biology and function. Pharmcol. Rev., 42, 355–402. Walovitch RC, Hill TC, Garrity ST, Cheesman EH, Burgess BA, O'Leary DH, Watson AD, Ganey MV, Morgan RA and Williams SJ (1989) Characterization of technetium-99m-L,L-ECD for brain perfusion imaging, Part 1: pharmacology of technetium-99m ECD in nonhuman primates. J. Nucl. Med., 30, 1892–1901. Walovitch RC, Franceschi M, Picard M, Cheesman EH, Hall KM, Makuch J, Watson MW, Zimmerman RE, Watson AD, Ganey MV, Williams SJ and Holman BL (1991) Metabolism of "Tc-L.L-ethyl cysteinate dimer in healthy volunteers. Neuropharmacology, 30, 283-292. Wester DW, Coveney JR, Nosco DL, Robbins MS and Dean RT (1991) Synthesis, characterization and myocardial uptake of cationic bis(arene)technetium(I) complexes. J. Med. Chem., 34, 3284–3290. Wilhelm AJ, Mijnhout GS and Franssen EJF (1999) Radiopharmaceuticals in sentinel lymph-node detection - an overview. Eur. J. Nucl. Med., 26(suppl.), S36-S42. Willerson JT, Parkey RW, Bonte FJ, Meyer SL, Atkins JM and Stokely EM (1975) Technetium stannous pyrophosphate myocardial scintigrams in patients with chest pain of varying etiology. Circulation, 51, 1046–1052. Zhen W, Han H, Anguiano M, Lemere CA, Cho C-G and Lansbury PT Jr (1999) Synthesis and amyloid binding properties of rhenium complexes: preliminary progress toward a reagent for SPECT imaging of Alzheimer's disease brain. J. Med. Chem., 42, 2805–2815.

11. CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS RONALD E. WEINERA AND MATHEW L. THAKUR8 A

Division of Nuclear Medicine, University of Connecticut Health Center, Farmington, CT 06030–2804, 06030-2804, BBDepartment Departmem of Radiology, Thomas Jefferson University Hospital, Philadelphia, PA 1910, USA.

INTRODUCTION Gallium and Indium, group IIIB of the periodic table, have several medically useful radionuclides, which have made extensive contributions in both the diagnosis and therapy of disease. The most useful among them are 67Ga and 111In. A variety of factors contribute to the utilization of these nuclides. The first and foremost are the useful physical characteristics of the radionuclide. These characteristics have prompted investigators to combine the nuclides with molecules that direct the nuclides to their intended target to identify some significant clinical conditions. In this regard, gallium-67 has a long and curious history (Hayes, 1978). In the late 1940's, gallium metal was suggested as reactor coolant in nuclear submarines because of its low melting point, 30-C and high boiling point, 19832C. Dudley and co-workers while investigating the toxicity of gallium (Dudley & Garzoli, 1948; Dudley et al., 1949) found that 72Ga localized in bone. They suggested that this nuclide might be useful for treating bone tumors. Subsequently Bruner, Brucer and co-workers at Oak Ridge National Laboratory extended these studies in humans (Brucer et al., 1953a; Bruner et al., 1953b). They concluded that 67Ga might be a better radioisotope since 72Ga has a short T 1/2 (14 hr) and was only available with low specific activity. Gallium-67 with a longer T 1/2 (Table 1) could easily be made carrierfree. However, imaging equipment at that time was not sophisticated enough to produce a good quality clinical study with this radioisotope. The development of the 68Ge/68Ga generator, in the early 60's, rekindled interest in radiogallium for bone tumor localization. However, this did not last long. Gallium-68 was discarded due to the concern about toxicity because of the amount of carrier gallium required for bone localization and the high radiation dose due to its annihilation photons (Table 1). In the early 70's, 67Ga reemerged since its characteristics were better suited to the newly developed instrument, the gamma camera and the lower amount of carrier gallium that was added to obtain bone localization. Clinical studies were initiated with carrier-free 67Ga that were to be followed with carrier-added 67Ga in the same patients(Edwards & Hayes, 1969). Early on in this project, the carrier-free nuclide was found unexpectedly to localize in certain soft tissue tumors and a few years later in inflammatory processes (Lavender et al., 1971). These two uses for 67Ga continue to this day.

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly, ©2003 John Wiley & Sons, Ltd

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364

Table 1. Important Nuclear Characteristics for Gallium Isotopesa 68

Characteristic

67,

y photon energy (keV)

93 184

300

511(p + )

%photons/disintegration

38 24

22

178

electrons (keV)

84, 92

1900(P+)

4153(P + )

half-life

78 h

68m

9.5 h

Ga

Ga

66,

'Ga 1039 2750

114

Decay

ECto 67 Zn

10 % EC to 68Zn 90% B*

43% EC to 66Zn 57% B*

Production method

68

68

63

Zn(p, 2n)67Ga

Ge daughter

37

23

Cu(o, n)66Ga

66

Zn(a, 2n)68Ge

Target abundance Contaminant

(18%) 66

(28%)

65

Ga,

68

Zn

Beam energy (MeV)

12–22

Target yield

55.5

Ge

12-22

(69%) 67

Ga

8-12

26

(MBq/jiA-hr) [Ar]3d104s24p1

Electron structure

"compiled from Zweit et al., 1987; Lederer & Shirley 1978a; Thakur, 1977 & Kowalsky &Perry, 1987a. The uses of111 In chloride were first investigated in animals in the late 1960's (Goodwin et al., 1969; Hunter & DeKock, 1969). In the early 70's,

111

In-DTPA was developed for cisternography and is still used for this

procedure today (Goodwin et al., 1975). In 1975, McAfee and Thakur (McAfee & Thakur, 1975) developed a technique to label blood cells with this nuclide. This led to the development of 111In-WBC as an agent for the detection of acute inflammation/infection. commercially available in the late 80's. radionuclide characteristics, four

This agent (111In-8-hydroxyquinoline [oxine]) became

In the early 90's, because of its appropriate chemical and

111

In-based agents were approved for human use. These agents included;

three antibodies and one peptide. Indium-111 labeled OncoScint was the first murine monoclonal antibody

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

365

that was approved by the FDA (January, 93). This was for the diagnosis of the recurrence of ovarian or colorectal cancer. lllIn-ProstaScint, another murine monoclonal antibody was approved for the diagnosis of prostate cancer recurrence, and 111In labeled MyoScint, a monoclonal Fab antibody fragment was approved for heart infarct detection. Lastly, 111In labeled OctreoScan, the first approved peptide-based agent, is used for the diagnosis and treatment of neuroendrocrine tumors. These two nuclides share a number of characteristics since indium follows gallium in group IIIB. They are both extensively hydrolyzed at physiological pH and are the most stable in the aqueous solutions in the +3 oxidation state (Cotton & Wilkinson, 1988). In addition, the characteristics of these nuclides have been compared to Fe3+ mainly because of the importance of the iron transport protein in the blood, transferrin (TF). Both 111In and 67Ga bind to this protein with high affinity (Harris et al, 1994; Harris & Pecoraro, 1983). Thus, this protein has an important effect on the biodistribution of these radionuclides. TF binding to these radionuclides must be taken into account when designing new radiopharmaceuticals. During the past quarter of a century numerous compounds have been synthesized that utilize the isotopes of gallium and indium for potential diagnostic and therapeutic applications (Reichart et al,, 1999). We will concentrate mostly on the chemistry of these nuclides and on those compounds of gallium and indium radionuclides that have shown direct clinical applicability. GALLIUM CHEMISTRY Gallium, a group IIIB element between Al and In, has a filled d shell and three electrons in its outer shell (Table 1). Its aqueous chemistry is dominated by its ability to form strong complexes with the OH" ion. The fully hydrated (hexaaquo) Ga3+ ion is only stable under acidic conditions. Once the pH is raised above 3, Ga begins to form insoluble Ga(OH)3 (Baes and Messmer, 1976). However, gallium is amphoteric. As the pH is raised further, the ion re-dissolves due to the solubility of [Ga(OH)4]~, gallate. A variety of OH intermediates are formed as a function of pH. The total concentration of gallium in solution is the sum of the hexaaquo ion plus all of the different hydroxo species, [Ga(OH)n ]3-n and any ligand-bound species The concentration of each of these species at a unique pH can be determined from the equations given below (Harris & Pecoraro, 1983) where [Ga]T is the total concentration of Ga in solution, the a's are pH dependent functions that account for various protonated and hydrolyzed forms of the species within the brackets (for example, OOH ), and pn is the formation constant for the individual hydrolysis species. The best estimates for individual formation constants are log Pi = -2.9, log p? - -6.6 and log P3= -11.0 and log p4 = -16.6 (Baes & Messmer, 1976). [GaJ T = ctOH[Ga] + aGaL[GaL] [L] T =a L [L] + aGaL[GaL] (XOH ^ 1 + Pi/[H] + P2/[H]2+ P3/[H]3 + P4/[H]4 [Ga] + nH 2O = [Ga(OH)n] 3-n + n [H] and

1-1 1-2 1 -3 1 -4

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HANDBOOK OF RADIOPHARMACEUTICALS

pn = [Ga(OH)n][H]n/[Ga]

1-5

Using these individual formation constants, the OQH can be calculated to be 1.02 x 1013 at pH 7.4. From this we can estimate that the distribution of gallium hydrolysis species is mostly gallate, [Ga(OH)4] (98%) and some Ga(OH)3 (1.6%). Because these hydrolysis species are in equilibrium, the solubility of the gallate is limited by the solubility of Ga(OH)3 whose minimum solubility is ~40 nM. Thus, the maximum total Ga concentration in solution at pH 7.4 has been calculated to be 2.5 jiM (Harris & Pecoraro, 1983). For nocarrier-added preparations of 67Ga, a concentration of 740 MBq/mL - 50 nM or for 68Ga a concentration of 3.7 GBq/mL = 40 nM. These calculations would indicate that gallium precipitation should not be a problem. However, citrate ion, a weak chelating agent (Table 2) is added for radiopharmaceutical preparation to prevent precipitation at neutral pH. It is possible that stable Ga is present in these preparations that is below the detection limit of 0.1 ^iM. Any non-radioactive Ga present in the target material would be separated along with the

67

Ga.

If other metal ions are present, particularly iron, co-precipitation at neutral pH is

possible. Table 2. pM and KML Values for Various Gallium Chelating Agents8 pMb

K ML C

DTPA

22.8

23.3

EDTA

21.7

21.7

DOTA

—-

21.33

TF(K,)

21.3

20.3

TF(K2)

20.3

19.3

NTA

19.04

16.2

OH^1

19.01

39.4

Citrate

—-

10.02

"compiled from Harris & Pecoraro, 1983; Clark & Martell, 1991 b

pM= -log[Ga(H2O)6], pM calculated for 1 uM Ga, 10 p.M ligand, and 27 mM carbonate, pH 7.4

C d

K ML = [ML]/[M][L]

Ga(OH)4

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

367

The importance of the hydrolysis of Ga3+ can be seen when the affinity of OH" is compared to a variety of other chelates. Table 2 shows the standard stability constants, KML and a more useful parameter pM (-log [M]) values. From equation 1-1, the pM value is the concentration of the uncomplexed hexaaquo gallium ion and is calculated under a specific set of conditions. The pM value takes into account the ligand basicity, metal ion hydrolysis, metal-ligand stoichiometry, dilution effects and other conditions. The larger the pM value, the better is the ligand for Ga3+. This value provides a direct head-to-head comparison among the various dictating agents under physiological conditions. Under these conditions the iron transport protein in the blood TF has a stability constant and pM comparable to diethylenetriaminepentaacetic acid (DTPA) and ethytenediaminetetraacetie acid (EDTA). The OH ion also has a relatively high pM value, only one order of magnitude lower than one of the TF binding sites. This suggests that if the concentration of TF was reduced an order of magnitude, [Ga(OH)]4~ would be thermodynamically favored. A computer simulation demonstrated that under normal conditions in the blood >99% of 67Ga is TF-bound (Jackson & Byrne, 1996). A reduction in the concentration of available TF sites increase the gallate concentration. This would increase gallate kidney excretion and could dramatically alter the biodistribution. While pM values are important, equally important is the speed with which this Ga transfer occurs. Binding experiments with TF suggest a rapid (-10-40 min) Ga equilibration (Harris & Pecoraro, 1983). Thus Ga could easily shift from TF to gallate. The chelate chemistry of gallium is dominated by ligands that contain nitrogen and oxygen as the electron donors. This is because gallium is classified as a hard acid in the hard acid soft base (HASB) system (Martell & Hancock, 1996). Hard acids prefer hard bases, highly ionic non-polarizable Lewis bases (proton acceptors). Gallium ion usually forms complexes with 4 to 6 coordination but not higher. Gallium ion shares chemical characteristics with the Fe3+ ion. However, gallium is slightly smaller, a slightly weaker Lewis acid (proton donor) and a bit softer than Fe (higher affinity for the softer RS-, Table 3). This similarity with the Fe ion is important for radiopharmaceutical development since iron is an essential in vivo element and the human body contains a wide variety of iron binding protein, the most notable is TF (see above). Table 3. Comparison of Properties for Ga3+, In 3+ , and Fe3+a ^ 3+

Ga

ionic radius

62 b

In3+

Fe3+

80

65

log K! (OH') c

111.3

10.0

11.8

logK 1 (NH 3 )

4.1d

4.0d

3.8d

logK,(RS-)

8.7

9.1

8.6d

a

Martell & Hancock, 1996 values in pm C K1=[ML]/[M][L] d estimated value

b

368

HANDBOOK OF RADIOPHARMACEUTICALS

There are, however, important chemical differences between these two ions, which lead to quite different biodistributions (Weiner, 19%). In this environment and at trace levels, 67Ga is soluble whereas iron is not For example, at the usual initial blood level of 74 Bq/mL (222 MBq/3000 mL plasma), the gallium concentration would be 0.050 nM. This is well below the solubility limit for gallium (see above). In contrast at pH 7.4, Fe(OH)3 has a Ksol ~ 10-38 M and iron tends to polymerize of the form FeOOH (Baes & Messmer, 1976). This limits the solubility of Fe3+ to ~10-l2 M. Iron requires proteins or other chelating agents for in vivo transport. For iron to be absorbed in the GI tract and eventually incorporated into hemoglobin or the cytochrome enzyme system requires iron to cycle between its two physiologic stable states, Fe2+ and Fe3+ (Weiner, 19%). In contrast, Ga3+ is the only stable state for gallium under physiological conditions. The oxidation potentials for each of these ions shown below demonstrate that the ferrous ion is favored while the gallous ion is not (Latimer, 1952). Ga3+ + e -4 Ga2+ E = -0.65; Fe3+ + e -> Fe2+ E = 0.771

1–6

Thus for iron in vivo, oxidizing agents, which are readily available, can recycle the Fe2+. This is very unlikely for gallium. Noujaim and co-workers showed that [59Fe]ferric ions are incorporated into RBC in humans but 67 Ga is not, providing support for this suggestion (Logan et al., 1981). RADIONUCLIDE'S PHYSICAL CHARACTERISTICS There are three radioisotopes of gallium suitable for imaging with present day equipment (Table 1). Among them 67Ga is the most utilized, due to its ability to identify both inflammations and soft tissue tumors (Alazraki, 1995; Neumann & McAfee, 1995; Neumann et al., 1995) and 66Ga is the least. There is interest in 68 Ga because it is a positron emitter, and thus useful in positron emission tomography (PET) or coincidence detecting gamma cameras. In addition, a more important aspect of this nuclide is that it is generator produced with a very long-lived parent. This nuclide could potentially fulfill a role similar to the 99Mo/99mTc generator providing a ready supply of 68Ga for the preparation of various radiopharmaceuticals. As yet no commercially available radiopharmaceuticals have been developed for this nuclide. Gallium-66, another positron emitter, is cyclotron produced with a medium T1/2 and moderate positron efficiency. This radioisotope has generated relatively little interest. While clinical usefulness is extremely important, ease of distribution and the cost is also an essential component for widespread use. Gallium-67 is cyclotron produced and has a long enough T1/2 so it can be made at a central location and readily distributed throughout a wide geographical area (Table 1). The nuclide can be shipped to users, radiopharmacies or hospitals, without significant losses of radioactivity due to decay. The loss due to decay is ~l%/hr. This means to achieve the desired delivered radiopharmaceutical dose the activity must start out about 15-25% greater, which is acceptable. With the proliferation of radiopharmacies that serve numerous hospitals, this nuclide can be stocked. Thus, a hospital could order and receive a 67Ga dose the same day.

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

369

The other factors that contribute to the cost are; the rate of their production, the efficiencies of their separation and the ease of the process required to convert them into desired radiopharmaceuticals. Table 1 shows that this nuclide is produced by charged particle bombardment of an enriched 68Zn in a cyclotron (Thakur, 1977). To isolate the 67Ga, the target is dissolved in HC1, the Ga extracted with isopropyl ether, back extracted from the ether into HC1, which is then evaporated to dryness. Citric acid is added to prevent hydrolysis; the pH is adjusted to near neutral and finally the 67Ga citrate solution is sterilized. The need for an enriched zinc target increases the cost because expensive procedures are required for zinc isotope separation. The energy of the bombarding protons is not unusually high and the bombardment can be carried out in moderately sized cyclotron. The rate of production is relatively high (Table 1). This all leads to very moderate, present-day price for 67Ga in the range of $60-80/6 mCi, a dose for an adult human. Where 67Ga has gamma photons that can be detected by the gamma camera but there are some physical characteristics that make this nuclide less than optimal for imaging. Gallium-67 probably has the worst characteristics of all of the gallium and indium radioisotopes (Tables 1 and 4). When 67Ga decays, less than 40 gamma photons are produced per 100 disintegrations with 93 keV energy. That is, in the other 60 disintegrations, 67Ga decays directly to the ground state of 67Zn without yielding a photon. The other higher energy gamma photons of 67Ga are even less efficient. The useful photon disintegrations for 1 1 1 In are greater than 90%. Thus, if we compare just photon efficiencies for imaging, 67Ga would need to be taken up twice as much by the target tissue to be comparable to 111In. Gallium-68 is a high efficiency P+ emitter (Table 1). This nuclide is produced by a very long-lived (275 d) 68 Ge parent. This is a big advantage because it provides 68Ga for up to 1–2 years. Also, the parent can be easily produced by a bombardment of an enriched Zn target at moderate particle energy. The problem has been in developing a generator that could provide the 68Ga in an easily usable form with a low amount of contaminating parent 68Ge. Welch and coworkers (McElvany et al., 1982; McElvany et al., 1984) have compared a number of generators. One using a tin dioxide matrix and elution with 1 M HCL was judged the best (Loc'h et al,, 1980). The yield was high 75-80% with low breakthrough of the parent, 0.0002 % of the column activity. Metallic containments were very low except for Sn and it was convenient to use for radiopharmaceutical preparation. LABELED RADIOPHARMACEUTICALS 67

PREPARATION AND QC Gallium-67 is cyclotron produced as described in "Radionuclide's Physical Characteristics". The preparation contains a high concentration of citrate, 0.12 M (Gallium citrate, 1992). The adult dose for chronic infections is 4 to 6 mCi and for tumor localization 6 tol0 mCi. To determine the radiochemical purity (RCP), the sample is spotted on Whatman Paper No. 1 using a 1:2:4 mixture of pyridine, ethanol and water as the solvent, The main contaminants are Ga(OH)3 Rf = 0.0 and Ga(OH)4, Rf = 0.6. Gallium citrate migrates to Rf = 0.6.

370

HANDBOOK OF RADIOPHARMACEUTICALS

INDICATION FOR SCINTIGRAPHY For many years 67Ga citrate was used for the detection of both acute and chronic abscesses and inflammatory processes. However, with the development of 111In-oxine or "Tc-HMPAO labeled white blood cells [111In or 99mTc-WBC] (see section 3.4), 67Ga is now used mostly to identify chronic inflammatory processes in diseases such as sarcoidosis, systemic lupus erythematosus and Sjogren's syndrome (Alazraki, 1995; Neumann & McAfee, 1995). This nuclide is also used to detect inflammations in patients where WBC function is impaired such as Pneumocystis Carinii Pneumonia, the major cause of death in AIDS patients. This radiopharmaceutical is the choice for radionuclide imaging of a number of soft tissue tumors (Neumann et al., 1995). These include the detection and staging of Hodgkin's disease, non-Hodgkin's lymphomas, lung cancer, hepatoma, and melanoma. Probably more important than just identifying the lesion is the ability of 67 Ga to determine response to therapeutic intervention. In contrast to radiologic examinations such as computed tomography, which detect morphological changes, 67Ga can differentiate recurrent viable tumor compared to scars caused by the chemo- or radiation therapy. In this manner, 67Ga can determine response to chemotherapy and detect recurrent tumor when the question is tumor vs scar (Kaplan, 1990). NORMAL DISTRIBUTION AND RADIATION DOSIMETRY After 67Ga citrate is injected normally 99% of the activity binds to the TF and a small percent of non-protein bound 67Ga (as gallate) remains in equilibrium. Normally, 2/3 of this TF circulates in the apo-(iron free) form. For the normal concentration of unsaturated TF (40 uM) in the blood, the gallate concentration is only 1 % of the total activity injected (Weiner et al., 1992). Because of the high pM of [Ga(OH)4] (Table 1) 67Ga can easily shift between TF and gallate depending on the effective TF concentration (Harris & Pecoraro. 1983). For example, if an increase in iron-saturated TF reduced the concentration of unsaturated TF from 40 to 5 pM, this would increase circulating gallate concentration to 7%. This would increase the amount of activity that passes out in the urine. This easy shift between [Ga(OH)4] and TF also shows up in the wide spread in vivo distribution of 67Ga. The volume of distribution (Vd) for the radionuclide is 23 L which implies that some 67Ga is distributed in the body water (extracellular fluid = 12 L; total body water = 41 L). Since 67Ga is normally tightly TF-bound it is excreted very slowly. For example, after 7 days in humans, only ~26% of the injected activity has been lost in the urine and only 9% in stool (Gallium citrate, 1992). The radioactivity is initially distributed in the liver (2.8% of injected dose/kg normalized to 70 kg man), spleen (4.1%), kidney (2.7%), and bone (2.6%), which remained unchanged (Nelson et al., 1972). However during the next few hours, a large quantity of radioactivity appears in the bowel. This obscures lesions in this anatomical region. The radiation dose for each 185 MBq in cGy is; whole body = 1.3, upper large intestine = 4.5, lower large intestine = 4.5, bone marrow = 2.9 (Gallium citrate, 1992). LOCALIZATION MECHANISM The localization mechanism of 67Ga both in tumors and abscess has been studied for more than three decades since its clinical utility was demonstrated. The preponderance of evidence suggests that transferrin (TF) and its receptors on the tumor cell surface are the most critical factor in uptake of 67Ga in tumor cells (Weiner & Thakur, 1995; Weiner, 1996 and references therein). In the 70's, it was shown that TF due to its iron-binding

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

371

ability stimulated 67Ga incorporation into cultured tumor cells. Larson and co-workers extended this hypothesis to specifically include the expression of TF receptors (TFR) on cancer cells (Larson et al., 1980). They proposed "a tumor-associated TF receptor is the functional unit responsible for the affinity of gallium for certain neoplasms" and provided data from both in vivo and in vitro model systems to support the hypothesis. The reason underlying this proposal was that at normal concentration of Fe-TF (complex of Fe and TF) in the blood, the TFR are saturated with Fe-TF. Thus to increase iron uptake, cells must upregulate the number of TFR. Iron is required for several cellular processes but TFR are regulated mainly to meet specific iron needs of DNA synthesis. The specific target for this iron is ribonucleotide reductase (RR), a non-heme iron-requiring enzyme. RR catalyzes the first rate-limiting step in DNA synthesis, converting ribonCicleotides to deoxyribonucleotides. In rapidly proliferating tumor cells with a high level of DNA synthesis, upregulation of the surface TFR would be expected. As an unintended consequence, this would promote increased 67Ga uptake. The importance of 67Ga scintigraphy as a diagnostic tool lies in its ability to both identify tumor and to differentiate viable tumors from scar after chemo- or radiotherapy. Additional evidence demonstrates the connection between "Ga uptake and cell viability and DNA synthesis (Weiner, 1996 and references therein). Both 67Ga and l8F-fluorodeoxyglucose uptake in experimental tumors had a parallel decline after chemotherapy and radiotherapy. Inhibiting tumor adenosine triphosphate (ATP) production causes a similar decline in 67Ga incorporation suggesting that 67Ga uptake correlated with cellular metabolism. The response to radiotherapy was monitored in a tumor model using metabolic tracers for glucose metabolism; DNA, RNA and protein synthesis and the uptake of these tracers were compared to 67Ga citrate. While 67Ga could not detect early response to treatment, it closely followed the diminution in glucose metabolism. The DNA synthesis indicator and 67Ga could differentiate between tumor with small foci of recurrence and non-viable tumor, whereas the glucose metabolic indicator could not. Non-radioactive gallium has been shown to interfere with DNA synthesis by the specific inhibition of ribonucleotide reductase.

CLINICAL CONDITIONS THAT ALTER THE BIODISTRIBUTION Since TF plays a pivotal role in delivering 67Ga to normal tissue, any condition that influences iron TF saturation can affect the biodistribution (Weiner, 1996 and references therein). Data from animal models and computer modeling of Ga species in blood paralleled very closely to what was observed in the few patient studies that had been reported. When the number of 67Ga binding sites on TF was reduced in a variety of animals either by iron injections, congenital lack of TF, irradiation or chemotherapy, the normal tissue (liver, spleen, bone marrow) uptake of 67Ga was depressed, whole body excretion was enhanced and 67Ga in bone was unaffected or increased. When intramuscular injections of iron were given to patients near the 67Ga injection time, to saturate their TF, blood 67Ga activity was significantly diminished but rebounded as the saturated TF was catabolized.

372

HANDBOOK OF RADIOPHARMACEUTICALS

Figure 1: A. anterior and B. posterior 24-hr images of patient with suspected fever of undetermined origin injected with 3.4 mCi of 67Ga. Patient with acute lymphocytic leukemia. Liver and spleen are absent on image. Increased activity in skeleton, particularly the joints, surgical scar and bladder are seen on the anterior image. Also increased bilateral kidney activity is visible on posterior image. Bone activity was also increased, tissue activity reduced and GI excretion was reduced. Individuals who had repeated blood transfusions that saturated their TF with iron had similar67Ga distributions. A typical gamma camera image had little or no apparent activity in the liver and spleen and high activity in bone and the kidneys (Fig. 1). Chemotherapy that likely increases TF saturation yielded comparable scintigrams.

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACLUTICALS

373

68

Ga

PREPARATION AND QC Gallium-68 is produced by a generator as previously described. A generator for research purposes only is commercially available from NEN Life Science Products, Boston MA. The matrix for this generator is alumina (Al2O3) and it is eluted with a 0.005 M EDTA solution as the solvent. The eluent is tested for 68Ge breakthrough i.e., parent activity in the elution. If the 68Ga-citrate is prepared then the 67Ga procedure would be used to determine RCP. INDICATIONS FOR SCINTIGRAPHY The short T1/2, of 68Ga mandates that this radionuclide be used where the target uptake is very rapid. In addition, target residence time should allow imaging to be completed within ~ 45-60 min. Galliurn-68 citrate has been used in patients to determine the extent of lung injury (Reichart et al., 1999). Since the nuclide binds immediately to TF, it permits the determination of the amount of TF leakage into the lung. Positron emission tomography allows the quantitation of lung uptake and thus the extent of lung injury can be estimated. Experimental animal studies have generally been limited to the development of myocardial and brain imaging agents. A number of 68Ga agents have been synthesized but none as yet have progressed to clinical studies. NORMAL LOCALIZATION AND RADIATION DOSIMETRY The biodistribution of 68Ga citrate is, of course, identical to 67Ga citrate. The only difference is that the dose to the patient is much lower primarily due to the much shorter T1/2 of 68Ga. The dosimetry in cGy/185 MBq is; whole body = 0.26, lower large intestine = 0.47, upper large intestine = 1.15, bone marrow = 0.5 (Kowalsky & Perry, 1987a). INDIUM CHEMISTRY Since indium is also a group IIIB element below Ga and has a filled d shell and three electrons in its outer shell, it shares many features with Ga (Table 4). Indium exists as the hexaaquo ion only at acid pH and also undergoes extensive hydrolysis at pH 3.4 and above, if not chelated. However, indium is much less amphoteric compared to Ga. The soluble [In(OH)4]", does not start forming until pH 7.4. In contrast, almost 100% of the Ga is in the form of the gallate at this pH. The concentration of each of these hydrolysis species at unique pH can be determined from the equations below. [In] T = aOH[In] + a InL [InL] [L] T =(x L [L] + a lnL [InL] a OH - 1 + p,/[H] + P2/[H]2+ (33/[H]3 + pY[H]4 [In] + nH 2 O = [In(OH)n] 3"" + n [H] and pn = [In(OH)n][H]n/[In]

1 -7 1-8 1-9 1-10 1-1 I

The parameters above have been defined previously for Ga. The best estimates for the formation constants are log p, - -4.3, log (32 - -9.4, log p3 = -13.9 and log p4 - -23.4, (Harris et al., 1994). The last two have been estimated at 0.1 M ionic strength and therefore carry more uncertainty. This uncertainty would influence the percent of the species formed at each pH. From these values we can estimate the concentration

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HANDBOOK OF RADIOPHARMACEUTICALS

of the various monoatomic species. At trace values, multiple atomic species are unlikely. We can calculate the value for a0H as we did for Ga. Thus at pH 7.4, the distribution of indium hydrolyzed species is mostly the neutral hydroxide, [In(OH)3] (99.1%) and very little In(OH)4 (0.9%). In contrast to Ga, the solubility of indium is limited by the solubility of the neutral In(OH)3 whose minimum solubility is ~50 nM (Baes & Messmer, 1976). For no-carrier-added preparations of 111In, a concentration at 740 MBq/mL = 400 nM, for no-carrier-added preparations 114m In, a concentration of 37 MBq/mL = 350 nM and freshly eluted 113mIn, a concentration of 3.7 MBq/mL = 50 nM. Thus the concern of hydrolyzed species is much greater for 111In than for 67Ga. Indium-111 is usually shipped as the chloride salt in a moderate concentration of HC1 (~0.05 M). Table 4. Important Nuclear Characteristics for Indium Isotopesa 111

114m

In

Characteristic

113m

In

In

Y photon energy (keV)

171

245

192

558

724

393

%photons/disint

91

94

17

3.5

3.5

64

electrons (keV)

0.6b 2.4, 25.4 22.3, 19.2

1985c(114In)

Half-life

67.4 h

50 d 100m

Decay

EC to 111Cd

ITto 114 In, 3 – to" 4 Sn

ITto" 3 In

Production method

112

" 4 Cd(p,n)" 4m In

"3Sn

Cd(p,2n) 111 In

daughter Cyclotron

"2Sn(n, y)"3Sn

Target abundance

(24%)

(28%)

Contaminant

"4mln

Beam energy (MeV)

12-22

6.5-12.6 MeV

Target yield (MBq/uA-hr) Electron structure

18.5

0.8

(1%)

[Kr]4d'°5s25p

a

compiled from Tolmachev et al., 2000; Kowalsky & Perry, 1987b; Silvester, 1978; Indium-111 Chloride, 1995; Lederer & Shirely, 1978b and Thakur, 1977 b Auger electrons c maximum energy The importance of the hydrolysis of In3+ can be seen when the affinity of the OH ion is compared to a variety of other chelates. Table 6 shows the standard stability constants, KML and pM values. The OH ion also has a relatively high pM value, only one order of magnitude lower than one of the TF binding sites. This

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

375

suggests that 111InCl3 would readily bind to TF when injected. However, if the TF concentration was reduced an order of magnitude, [In(OH) 3] would now be thermodynamically favored and colloidal indium could form. Table 6 also shows that both acetate and citrate ions are very weak chelates particularly compared to DTPA although the pM values are not available to make a better comparison. This low affinity combined with relatively high pKa values for acetate (4.7) and citrate (3.06, 4.74, and 5.4) provide a means to keep 111In in solution at higher pH's, 5-6. The pKa is the pH where there are equal concentrations of the acid and base species. Only fully deprotonated (base) species binds to the metal ion. The higher pH is particularly important in binding 111In to DTPA coupled to antibodies. Antibodies, like many proteins, are particularly sensitive and could precipitate or denature in acidic conditions. Thus, high concentrations of either acetate or citrate ions are routinely used to raise the pH of the commercial acidic 111In solution to allow translocation to DTPA conjugate. Table 5. Metal Transferrin Binding Constantsa log K 1M b log K2Mb

log UOH

Fe3+

21.44

20.34

-10

11.4

Ga3+

19.75

18.8

12.9

6.9

in3+

18.3

16.44

-8.3

-10.0

log K lef f

"Harris et al., 1994 Independent of bicarbonate concentration, K1M - [TF-M]/[TF][M], K2M - [TF-2M]/[TF-M][M] Under physiological conditions TF has a pM 7 orders of magnitude lower than DTPA. These values also suggest that 111In-DTPA complex should be exceedingly stable in the blood with little activity translocated to TF if the concentration of both were equal. This is why most antibodies and peptides that use 111In as a tracer are covalently coupled to DTPA. However, in vivo the concentration of these 111In labeled compounds is usually 5000-fold less or lower than the TF concentration when injected and could be much lower as time progresses. This partially negates the high pM of the DTPA. We must keep in mind that the pM values are conditional constants dependent on both pH and concentration. While pM values are important, equally important is the speed with which this 111In transfer occurs. Binding experiments with TF and other ligands suggest a very slow (~6-8 hr) indium equilibration (Harris et al, 1994; Welch & Welch, 1976). Thus 111In cannot easily shift from DTPA to TF even at high TF:DTPA ratios. Indium (IV) is also a hard acid in the HASB classification (Martell & Hancock, 1996). Thus indium (IV) prefers to bind to hard electron donors, nitrogen and oxygen. However, indium (IV) is much larger, a much weaker Lewis acid and softer than either Ga3+ or Fe3+ (highest affinity for RS", Table 3). Indium (IV) forms complexes with the usual 4 to 6 coordination but can also form complexes with 8 coordination (Martell & Hancock, 1996, see also Fig. 2). Table 5 shows that indium (IV) binds to TF with a lower affinity than either Ga3+ or Fe3+. The softer In3+ should form a weaker complex with the harder phenolate ion (tyrosinate).

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These ions are part of the TF metal ion binding groups (Harris et al., 1994). This lower affinity is contrary to several studies that have reported that 111In-TF is much more stable than 67Ga-TF. The more extensive hydrolysis of Ga compared to In is the likely reason for this difference. The OOH values computed from equations 1-3, 1-9 and a similar one for Fe, demonstrate that indeed Ga is the most hydrolyzed metal ion (Table 6). In addition, if an effective affinity constant is computed, K1eff = K1M/OOH, the value for Ga is 3 orders of magnitude lower than In. Table 6. pM and KML Values for Various Indium Chelating Agentsa pM b

K ML

DTPA

25.9

29.0

DOTA

—-

23.90

EDTA

0.7

21.70

TF(2:1)

18.7

16.64

NTA

—

15.56

OH-c

17.7

36.9

Oxine

—-

13.3

Citrate

—

6.18

Acetate

—

3.50

a

from Harris et al., 1994; Pecoraro et al., 1982; Thompson, 1978; Reichart et al., 1999 and Welch & Welch ,1975. b Calculated for 1 uM In, 10 uM ligand, and 5 mM carbonate, pH 7.4 c In(OH)3 In an aqueous environment, the In3+ valence is the most stable, similar to Ga3+ (Welch & Welch, 1975). The potential for formation of this species is given in equation 1-12. The In 1+ can be formed in acidic solutions but equation 1-13 shows that it is not thermodynamically favored. In^In 3 + + 3e In 1 + -*In 3 + +2e

E = 0.342 E = 0.425

1-12 1-13

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

377

Figure 2. Ortep view of the structure Na2In(DTPA)-7H2O. Reprinted by permission of the Society of Nuclear Medicine from: Macke HR, et al. The Molecular Structure of Indium DTPA. J. Nucl. Med. 1989; 30: 1235–1239 Figure 2.

RADIONUCLIDE'S PHYSICAL CHARACTERISTICS Indium also has three radioisotopes that can be used for imaging by present day equipment (Table 4). Of the three radionuclides, 111in is the one that is most widely utilized. A wide variety of radiopharmaceuticals have been developed using this nuclide. The most widely used is 111In-WBC. In addition, a number of clinical investigations are in progress in which antibodies and peptides labeled with 111In are the subject of therapeutic and diagnostic evaluations. On the other hand, 1114mIn has been used in a few clinical trials for therapeutic purposes (Sharma et al., 1997). The advantage of 114mIn is its ability to both deliver a radiation dose through its P particles and be used to determine the biodistribution. In the 70's, 113mIn was widely used because of its availability from a generator (Table 4). With the widespread availability of 111In, 113mIn is now little used. Similar to 67Ga, 111In is cyclotron produced with a long enough T1/2, so that it can be distributed over a wide geographical area (Table 4). 111 indium is produced with an energy range of protons similar to those needed for the production of 67Ga but the target need not be enriched (Lamb, 1982). This would imply that all things being equal 111in should have a lower per unit production cost. However, at present, primarily due to the relatively low clinical utilization of mIn nationally, 111In costs 4-5 times more than 67Ga. The number of different radiopharmaceuticals that use 111 In is much greater than, but the individual clinical utilization of these procedures is low. This may change with the recent introduction of a radiopharmaceutical to detec' prostate cancer, ProstaScint. This disease has very high prevalence, which could lead to greater 111In utilization as this radiopharmaceutical.

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HANDBOOK OF RADIOPHARMACEUTICALS

Indium-111 is purified from cadmium in a similar manner to the 67Ga purification. There are a number of cycles of extraction into ether and back extraction with HC1 to remove iron and other impurities. The final product is dissolved in sterile, pyrogen-free, 0.05 M HC1. As discussed earlier, its imaging characteristics are much better than 67Ga. Indium-111 has two photons with 90% or greater photon yield (Table 4). The only slight disadvantage is the two photons are somewhat higher energy than optimal since most gamma cameras are optimized for 140 keV. Indium-111 also possesses Auger electrons that have a very short penetration range [0.02-10 um] in tissue (cell diameter = 10 um) [Silvester, 1978]. This means that 111In can potentially be used for therapeutic applications if the complex containing the nuclide could be internalized in the target cell. This would allow the Auger electrons to interact with critical cellular function particularly the DNA while causing minimal toxicity to normal cells. Indium-114m is also cyclotron produced with a slightly lower range of proton energy (Table 4). Indium114m has a much longer T1/2. The advantage of this nuclide is that is has both an energetic 3 for therapy and a moderate energy photon, albeit with low abundance, for imaging. The energetic 3 has a long penetration in tissue, ~300–1,000 urn, thus can irradiate hundreds of cells. It could be used coupled to an antibody that targets tumors. Many tumors have low interior perfusion but still could be irradiated by this nuclide. The 7 photon allows accurate assessment of the radionuclide biodistribution. Indium-114m can be prepared relatively economically through a thermal diffusion separation from an enriched 114Cd target (Tolmachev et al., 2000). The 114mIn is separated from the Cd in an acid solution using a cation exchange resin with a 60% yield. Although a large number of 111In radiopharmaceuticals have been prepared and evaluated, only a few prominent clinically utilized compounds are discussed (Reichart et al., 1999). LABELED

RADIOPHARMACEUTICALS

INDIUM-111-OXINE LABELED WHITE BLOOD CELLS (111In-WBC) PREPARATION AND QC Indium-111-WBC are prepared by removing ~ 50 mL of the patients blood, isolating the white cell fraction from the plasma, incubating this fraction for ~15 min with 111 In-oxine to label the WBC at room temperature (Thakur et al., 1977a). The plasma is added back to cells and the cells centrifuged to remove non-bound activity. The cells are re-suspended in plasma and finally injected into the patient. The 111 In-oxine, is prepared by adding oxine in ethanol to 37-185 MBq [1-5 mCi] of 111In in acetate buffer pH 4.8- 5.5, and the resultant 111In-oxine is extracted in chloroform or methylene chloride. The organic layer is removed, solvent is evaporated, and complex dissolved in ethanol with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer containing 100 ug of tween 80. This solution is added to the cells. Figure 3 shows a schematic diagram of the labeling mechanism. Because three oxine molecules are bound to 111In, neutralizing the charge on the radionuclide, this makes the complex highly lipophilic (Fig. 4). Thus, the 111 In-oxine can intercalate into the cell's membrane and eventually pass into the cytoplasm (Choi & Hwang,

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS 1987). There 111In is translocated to proteins with higher 111In affinity (Thakur et al., 1977b). Finally, the oxine leaks out of the WBC. It is essential to remove the plasma before adding 111In-oxine for labeling because TF has a higher affinity for 111 In and a high concentration (40 jaM) in plasma (Table 5). However, once the cells are labeledand andthethe 111In is locked inside, plasma can be added to bind "free 111In" without a loss of labeling efficiency.

LABELING MEDIA

11

In-oxine

oxine

CYTOPLASM

11

In-oxine

oxine

Proteins

111

I

In-Proteins

Figure 3. Diagram of the labeling mechanism of white blood cells with 111In-oxine. The complex intercalates into the cell membrane, penetrates into the cytoplasm and there 111in is translocated to proteins with higher affinity for the radionuclide. The patient dose is limited to 18.5 MBq because of the high-absorbed dose 111In-WBC delivers to the spleen (see section "Normal Localization and Dosimetry"). The blood cells should be injected within 4 hr of removal from the patient; otherwise the ability of the WBC to migrate to inflammatory foci can be severely compromised. The viability of labeled cells after labeling is determined by a dye exclusion method. The cells are examined under a microscope for colored cells. Dye incorporation into the cell indicates membrane permeability, hence cell damage.

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HANDBOOK OF RADIOPHARMACEUTICALS

Figure 4. Ortep drawing of the structure for 111In-(8-hydroxyquinoline)3. Reprinted by permission of the Society of Nuclear Medicine from: Green MA, et al. The Molecular Structure of Indium Oxide. J. Nucl. Med. 1988; 29: 417–420 Figure 2.

INDICATIONS FOR SCINTIGRAPHY Since its introduction in 1977 (Thakur et al., 1977c) 111In-WBC have been successfully used to identify sites of acute inflammation, and infection (including osteomyelitis) (Preston, 1995). NORMAL LOCALIZATION AND DOSIMETRY After injection a large fraction of 111In is distributed equally in the liver and spleen [~30% each] (indium111-oxine, 1986). There is only transient pulmonary uptake. Activity is observed in the bone marrow. A portion of blood activity clears with a T1/2 of 2.8-5.5 hr while a small fraction has a longerT1/2of 64 to 116 hr. Very little of the radioactivity is eliminated from the body and release from the liver and spleen is also very slow. The absorbed dose for 18.5 MBq is for the whole body = 0.31 cGy, spleen - 13, liver =1.9, bone marrow = 1.3, testes = 0.01, and ovaries = 0.19.

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

381

Table 7 Parameters for Disorders of Destruction and Production of Plateletsa Thrombocytopenia Diagnosis

Normal ITP SLE Pre-splenectomy Post-splenectomy Infection Relapse Lymphosarcoma Hodgkin's Disease Consumptive Coagulopathy

Diagnosis

Disorders of Destruction Survival Count Days /pL

Effective Production X normal

250,000 16,500 46,200 6,900 190,000 468,000 7,800 32,300 59,200

9.9 0.14(2.3hr) 0.89 0.04 1.8 8.00 0.04(1 hr) 0.69 1.18

35,000 185,000 75,000 299,000 123,000 66,500 215,000 134,000 205,000

x1 5.3 2.1 8.5 3.5 1.9 6.1 3.8 5.9

51,300

1.03

77,200

2.2

Turnover Platelets pL/day

Effective Production X normal

Disorders of Production Count Survival /pL Days

Drug Marrow Damage Marrow Replacement by Fibrosis Marrow Replacement by Lymphoma a

Turnover Platelets pL/day

Compiled from Harker & Finch, 1969

16,900 15,000 12,300

8.0 5.0 8.0

3,100 0.1 9,900 0.3 5,100 0.1

382

HANDBOOK OF RADIOPHARMACEUTICALS

INDIUM-lll-OXINE LABELED PLATELETS (111In-Platelets) PREPARATION AND QC First, a low speed spin (150g for 10 min) is used to separate the platelet rich plasma (PRP) from the leukocytes and RBC's; the supernatant is removed containing the PRP (Rodrigues et al., 1999). Next, a harder spin (640g for 10 min) of the PRP pellets the platelets, the platelets are washed once to remove the TF-containing plasma, 18.5-37 MBq of 111 In-oxine is added, and cells incubated 15-20 min at room temperature. After the incubation, the platelet poor plasma (PPP) is added back, washed once with PPP, and the platelets injected. Platelet viability is determined by their ability to aggregate with stimulation, a normal function of these cells. Aggregation of labeled platelets is compared to unlabeled ones. The dose is limited to 18.5 MBq but only 1.85 to 5.55 MBq is used when a survival is performed. Recovery of platelets in the circulation immediately after injection can be determined by withdrawing blood at various times postinjection. The normal value is 50-60%. A lower value indicates that the percent of platelets is lost immediately, suggesting functionally impaired platelets. INDICATIONS FOR SCINTIGRAPHY The main indication is to differentiate production vs destruction defects in patients with thrombocytopenia (Louwes et al., 1999). Patients with destruction defects have shortened platelet survival (Table 7). In contrast, patients with production defects have normal survival. To determine the survival, blood is withdrawn at various times post- injection. The percent injected dose vs time is plotted to determine the T1/2 of the blood activity. Normal T1/2 = 7 ±2 days. This is not life span since 111In-oxine is a heterogeneous label, labeling both old and new platelets. NORMAL LOCALIZATION AND RADIATION DOSIMETRY After injection a large fraction of the 111In is distributed mostly in the spleen (38%) and some in the liver (13%) (Scheffel et al., 1982). Activity is observed in the bone marrow. Very little of the radioactivity is eliminated from the body and release from the liver and spleen is also very slow. The dosimetry is for the whole body 0.3 cGy/18.5 MBq, spleen= 16.75, liver=1.0, testes= 0.1, ovaries =0.2 and bone marrow= 0.5. INDIUM-111 LABELED PROSTASCINT (111In-ProstaScint) PREPARATION AND QC Indium-111-ProstaScint is prepared using a 2-vial system (ProstaScint, 19%). 185-222 MBq of 111 InCl 3 is added to vial 1 containing 0.5 M acetate buffer. The acetate buffer raises the solution pH (~5-6) and forms a weak 111In complex that prevents hydrolysis and possible precipitation. The contents of vial 1 is added to vial 2 containing 0.5 mg of antibody, and then incubated for 30 min at ambient temperature. In this process, the 111In is transchelated to the DTPA-antibody conjugate. Adding the 111InCl3 directly to the protein without the acetate buffer could hydrolyze the In and the HC1 could denature the antibody. This solution is filtered (0.22 um) prior to injection to remove any aggregates formed. The dose is 167–204 MBq and the preparation is injectable for 8 hr. To determine the RCP, the sample is mixed with DTPA (from a commercial kit) and incubated for a few minutes; the mixture is spotted on instant thin layer chromatography- silica gel (ITLC-

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS SG) media using saline as the solvent. Indium-111-ProstaScint remains at the origin and migrates to the solvent front (SF).

383 111

In-DTPA

INDICATIONS FOR SCINTIGRAPHY ProstaScint (originally designated 7E11-C5.3) is a whole IgG1, murine monoclonal antibody that was generated from human prostatic cell line. Presently it is believed that only broken or leaky cells take up the antibody since it is directed against the cytosolic portion of prostate specific membrane antigen (PSMA). Although the antigen is expressed on both primary and metastatic lesions, this antigen also has increased expression in benign prostate disease. Thus, the initial diagnosis must be confirmed by a tissue biopsy. The antibody containing a DTPA chelate is coupled specifically via a spacer molecule to the carbohydrate portion of the IgG and has the form, antibody-carbo-glycyl-tyrosyl-lysine-DTPA (Fig. 5). This is an improvement on the coupling procedure initiated by Hnatowich (Hnatowich, 1990). DTPA binding to the carbohydrate prevents its coupling to amino acids required for antigen-binding and decreasing the affinity. Furthermore, the spacer molecule positions the DTPA well away from the antibody minimizing any steric hindrance by the antibody that could reduce the 111In DTPA affinity. ProstaScint can be used to determine the extent of disease once the initial diagnosis is made (Quintana & Blend, 2000; Polascik et al., 1999; Manyak et al., 1999). If the disease is not confined to the prostate bed or nearby lymph nodes then hormonal therapy is the preferred form of treatment and not a radical prostatectomy. In this operation, the prostate and nearby lymph nodes are removed. ProstaScint can also be used to detect the extent of recurrence in the face of rising PSA after a radical prostatectomy(Murphy et al., 2000; Elgamal et al., 1998).

-s-s-

-s-s•s-s-

gly-tyr-lys

O H II H2-N-C-CH2

DTPA

Figure 5. Schematic drawing of the attachment of the 111In binding chelate, DTPA, to either ProstaScint or OncoScint. A peptide spacer is coupled to the carbohydrate of the antibody and then the amino terminal of the spacer is coupled to one of the carboxylic acids of DTPA.

384

HANDBOOK OF RADIOPHARMACEUTICALS

ProstaScint can detect so called "skip metastases" in abdomen, which have not been detected by other means. This test has a modest negative and positive predictive value of 50% and 70%, respectively (ProstaScint, 1996). However, it is much better than the standard radiology technique, computed tomography, which is very poor in detecting these lesions, previously identified. These are metastatic lesions that appear outside the prostate bed with the lymph nodes in the pelvic area not being involved. The prevalence of adverse reactions is minimal (4-5%). For the second infusion, however, 19% of patients were documented to have reactions.

Figure 6: A. anterior and B. posterior 144-hr (6-day) images of patient with prostate cancer injected with 5.3 mCi of 111In-ProstaScint. Normal uptake in the liver, spleen, heart and bone are easily seen on the anterior image. Modest kidney activity is observed on the posterior image. BIODISTRIBUTION AND RADIATION DOSIMETRY ProstaScint has a long T1/2 in the blood, 68 hr (ProstaScint, 1996). It is excreted very slowly, with a plasma clearance =35 mL/hr, Vd =37 mL/kg. After 72 hr, there is only 8% of the activity in the urine. The normal tissue distribution is liver, spleen, heart (blood vessels), bone marrow and some activity in kidney (Fig. 6). The dosimetry for the whole body is 2.9 cGy/185 MBq, spleen = 4.8, live r=10, and bone marrow = 3.6.

CHEMISTRY OF GALLIUM AND INDIUM RADIOPHARMACEUTICALS

385

INDIUM-111 LABELED ONCOSCINT PREPARATION AND QC Indium-111-OncoScint, like ProstaScint, is prepared also using a 2-vial system (OncoScint, 1992). 185-222 MBq of 111InCl3 is added to vial 1 containing 0.5 M acetate buffer. The contents of vial 1 is added to vial 2 containing 1 mg of antibody, and then incubated for 30 min at RT. This solution is filtered (0.22 pm) just prior to injection to insure sterility. The dose is 185 MBq (5 mCi), and the preparation is good for up to 8 hr. To determine the RCP, the sample is mixed with DTPA (from a non-radioactive clinical kit) and incubated for a few minutes. This mixture is spotted on ITLC-SG and eluted with saline. Indium-111-OncoScint remains at the origin and 111In-DTPA migrates to the SF. The DTPA chelates the non-bound 111In. The nonbound activity should form 111In(OH)3. This would also remain at the origin and obscure the actual percent bound. INDICATION FOR SCINTIGRAPHY This radiopharmaceutical contains a monoclonal antibody originally identified as B72.3 or CYT-103, which is directed against a tumor associated antigen called TAG-72 (OncoScint, 1992). TAG-72, a high MW, mucin-like glycoprotein, is expressed on 94% of colorectal adenocarcinomas and 100% of ovarian cancers tested. The 111In-binding site is DTPA covalently coupled to the antibody in a manner similar to ProstaScint (see Fig. 5). 111In-OncoScint, while not as useful for the initial diagnosis of these diseases but may provide an important adjunct in disease management and recurrence (Blend & Bhadkamkar, 1998; Pinkas et al., 1999). One-third of patients originally diagnosed with either of these cancers have recurrence within 18-24 months. A rising concentration of carcinoembryonic antigen (CEA) for colorectal cancer or CA125 for ovarian cancer in the blood is used as indicator. These antigens are readily shed from these tumor types but the blood test is sometimes unreliable. OncoScint scintigraphy is superior to other commonly used imaging modality, CT, in detecting disease sites in the pelvis, lymph nodes, bone, lung and brain but not in the liver. This is because the radionuclide accumulates in the liver excessively and nonspecifically. However, this radiopharmaceutical has a low negative predictive value (19%) and a negative image does not rule out the presence of disease (OncoScint, 1992). OncoScint is now approved for repeat usage. It was originally only approved for one time use because the radiopharmaceutical induced HAMA in 40% of the patients. HAMA causes rapid blood disappearance of subsequently injected OncoScint and interferes with CEA assay. It was determined that at 4 weeks post-injection, one-half of patients became HAMA negative. Thus prior to a second injection of OncoScint patients should be tested for HAMA. A HAMA of <50 ng/mL would not interfere with the study. Other adverse reactions e.g.; fever or chills, were experienced in 2-4% of the patients studied and only 0.7% experienced serious (anaphylaxis) reactions. NORMAL LOCALIZATION AND RADIATION DOSIMETRY After injection, 111In-OncoScint has a long terminal T1/2 in the blood of 56 hr (OncoScint, 1992). Since it is a large protein (150 kDa) it is initially distributed mainly in the plasma and small fraction in the extracellular space (Vd =3.8 L; plasma volume = 3 L). Whole body excretion is very slow, with the principle route of

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excretion via the kidney and the plasma clearance =50 mL/hr. In 72 hr, only 10% of the injected activity is eliminated. The normal biodistribution includes early localization in liver and spleen. Bone marrow activity appears at 24 hr and then persists, as does the activity in blood pool (due to long T1/2), in the heart and in major blood vessels. Faint kidney or bladder uptake may be seen on later images. The radiopharmaceutical dose is limited by the absorbed dose to the spleen, liver and bone marrow, which is 16, 15, and 12 (cGy/185 MBq) respectively (OncoScint, 1992). Other values are moderate; whole body 2.7, and ovaries 2.9. INDIUM-111 LABELED OCTREOSCAN (111In-OctreoScan) PREPARATION AND QC OctreoScan minimize non-specific indium binding) to a vial that contains 10 jig of pentetreotide and incubate is prepared by adding 111IMBq [3 mCi] of 111InCl3 (contains 3.5 pg FeCl3 to 30 min at RT (OctreoScan, 1994). A Sep-pak C18, mini-column is used to perform RCP test. The column is washed with methanol and water, and the sample is added to the column. A wash with water removes the unbound 111In and methanol wash elutes the 111In-OctreoScan with usually >90% RCP. The diagnostic dose is 185–222 MBq (2 vials of pentetreotide are used) and the preparation is good for 6 hr. For therapy the doses range from 3.7-7.4 GBq. INDICATIONS FOR SCINTIGRAPHY OctreoScan is an analog of somatostatin, octreotide, modified by adding an 111 In-binding group, DTPA (OctreoScan, 1994) [Figure 7]. Somatostatin is an endogenous peptide involved in the inhibition of growth hormones. 111In-OctreoScan is used to localize a wide variety of neuroendocrine tumors, e.g., carcinoids, islet cell tumors, paragangliomas, medullary thyroid cancers, pheochromocytomas, gastrinomas, and anterior pituitary adenomas (Krenning et al., 1993; Gibril et al., 1999; van Eijck et al., 1999). Studies are in DTPA-CO-NH-Phe-Cys

A

Phe

I S

\ D-Trp

S |

, I Lys

Thr(ol)-Cys —

HOOC

Figure 7. Schematic drawing of A. OctreoScan and B. diethylenetriaminetetraacetic acid (DTPA). Amino acids in bold are directly involved in somatostatin receptor binding. Amino acids are all L-isomers except where noted or an amino alcohol, Thr (ol). DTPA is coupled to the peptide via an amide bond and the methylene groups (CH2) are omitted for clarity.

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progress to determine if this radiopharmaceutical can also be used as a therapeutic agent for the treatment of these tumors when other treatments fail (Krenning et al., 1999; McCarthy et al., 2000; Cornelius et al., 1999). Figure 8 shows an example of a patient treated with high dose of 111In-OctreoScan.

Figure 8. Effects of radionuclide therapy in a patient with hepatic metastasis from a neuroendrocrine tumor. Patient received 7 doses for a total of 1.8 GBq 111In-OctreoScan. The left panel shows the planar image OctreoScan [anterior (left) and posterior (right)] in a patient treated on the therapeutic protocol. Multiple areas of increased uptake of the radioisotope, including the liver are evident. CT images from the pre(middle panel) and early post-treatment (right panel) scans demonstrate no significant change in the size of the tumors, but extensive central necrosis (dark areas). Dosimetry of a large representative lesion in the left lobe indicates that the estimated dose of radiation to this lesion is 1828 cGy. (From Drs John Murren, Eugene Cornelius and Irvin Modlin, Radioisotope Program at Yale with permission; Cornelius et al,, 1999)

NORMAL LOCALIZATION AND RADIATION DOSIMETRY This radiopharmaceutical is rapidly cleared from the blood. For example, at 10 min. only 33% of the injected dose remains in the blood. It is rapidly excreted into the urine (OctreoScan 1994). Renal excretion is the major excretion route. Twenty-five % of the injected radioactivity is present in the urine after 3 hr, 50% after 6 hr and 90% after 48 hr. Hepatobiliary excretion is minor, < 2% appears in the feces after 72 hr. After intravenous administration accumulation of activity is observed in the thyroid gland, pituitary gland, liver, spleen, kidney, and bladder. Gallbladder and intestinal activity are seen at later time points. At 4 hr postinjection, 2% of the injected dose is in the spleen, 1.9% in the liver, 7.2% in the kidneys. The dosimetry for a 222 MBq [6 mCi] dose is kidneys= 11 cGy, bladder wall = 6, ovaries = 1, bone marrow - 0.7, and spleen is the critical organ with 15. LOCALIZATION MECHANISM Somatostatin is a peptide hormone, which inhibits the release of growth hormone, insulin, glucagon and gastrin (Krenning et al., 1993). Somatostatin receptors (SSTR) have been identified on many cells of neuroendocrine origin e.g., anterior pituitary and the islet cells of the pancreas. More importantly, tumors of neuroendocrine origin have increased expression of these receptors. To enhance this potential, the hormone was modified to increase its extremely short blood T1/2, 2-4 min, and octreotide, an 8-peptide analogue (1400 Da), was the result. With the longer T1/2, localization and imaging of the radiolabeled octreotide was

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possible. There is little metabolism of 111In-OctreoScan. At 4 hr, the 111In is still bound to the intact peptide in the blood and only 10% of the total activity in the urine is nonpeptide bound. Lastly, there is a strong correlation between a positive image and an in vitro assay of SSTR in excised tumor tissue. This suggests that 111In-OctreoScan is an in vivo indicator of SSTR.

Figure 9. Photomicrograph of tumor tissue autoradiograph from a patient injected with 111In-OctreoScan. 111 In-OctreoScan A. bound to the plasma membrane (pm) B. internalized into the cell in or close to vacuoles, which are mingled among secretory granules, C. in the perinuclear area and D. in the nucleus (n). Arrows indicate the position of silver grains. Exposure 4 or 7 d, magnification A-C. X24,000 and D X36,000 bars = 500 nm. Reprinted by permission of the Society of Nuclear Medicine from: Janson ET, et al. Nuclear Localization of 111In after Intravenous Injection of [111 In-DTPA-D-Phel]-Octreotide in Patients with Neuroendocrine Tumors. J. Nucl. Med. 2000; 41: 1514–1518 Figure 4.

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For therapeutic purposes, the localization process after receptor binding is most important. In vitro data support that the

111

In-OctreoScan is internalized into cell and localized near the nucleus. Most recent data

that utilizes tumor tissue from patients injected with

111

In-OctreoScan shows results consistent with this

hypothesis (Tiensuu- Janson et al., 2000). Figure 9 shows a sequence of photomicrographs where

111

In-

OctreoScan is localized on the tumor membrane, cytoplasm and then finally inside the nucleus. Nuclear localization is essential for this therapeutic effect since the Auger electrons have short penetration distance and a double strand DNA break is the most lethal to the cell (Gardin et al., 1999).

DOTA-CO-NH-D-Phe-Cys

Tyr,

I

!

A

iI

^

!

Lys

Thr(ol)-Cys —Thr

>

n

rv-»^

COOH

Figure 10. Schematic drawing of the structures of A. DOTA coupled to a modified octreotide [tyr replaces phe in position 3] and B. tetraazacyclododecanetetraacetic acid (DOTA). Amino acids in bold are directly involved in somatostain receptor binding. Amino acids are all L-isomers except where noted or an amino alcohol, Thr (ol). DOTA is coupled to the peptide via an amide bond and the methylene groups (CH 2 ) are omitted for clarity. As an alternative therapeutic approach, modified octreotide has been labeled with another chelate, 1,4,7,10tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) (Figure 10). This chelate has a high affinity for both 111In (Table 5) and 90Y (log KML = 29).

90

allows for the possibility of so-called "crossfire".

Y is a high-energy (3" emitter (Maximum 2.3 MeV), which The particle range, 1000 |nm (~100 cells) allows the 90 Y-

DOTA-octreotide internalized or bound to the cell's surface to irradiate cells in the nearby vicinity. 90

downside of Y label is that this nuclide does not have a y photon for easy imaging. Thus, the derivative is used to estimate the biodistribution of the 90Y-DOTA-octreotide (Otte et al., 1997).

111

The

In labeled

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INDIUM-111 LABELED MYOSCINT (111In-MyoScint) PREPARATION AND QC Indium-111-MyoScint is prepared using a 2-vial system (MyoScint, 19%). The contents of vial 1 with 0.5 mg of antibody in phosphate buffer pH 6.5 is added to vial 2 containing 0.2 M citrate buffer pH 5 and mixed. Then 92.5 MBq [2.5 mCi] of 111InCl3 is added to vial 2, and incubated for at least 10 min at RT. The citrate buffer rapidly forms a weak 111In complex. This allows transchelation to the DTPA-antibody conjugate. Just before injection the solution is passed through a 0.22 fim filter. To perform RCP analysis, the sample is spotted on a strip of ITLC-SG and then eluted with 0.1 M citrate buffer pH 5.0. The unbound 111In is chelated by the citrate and migrates to the solvent front while the 111In-MyoScint remains at the origin. INDICATIONS FOR SCINTIGRAPHY MyoScint is an anti-myosin Fab fragment antibody, with an attached DTPA chelate (MyoScint, 19%). Myosin is the major intracellular muscle protein and is only exposed when the cell membrane is damaged. This protein is only soluble in high salt concentration and is not found in the blood after cell rupture. In contrast to 201Tl, 111In-MyoScint localizes in necrotic myocardial tissue. An infarct would appear, as hot spot and normal tissue would not be localized by this agent. Indium-111 -MyoScint would probably not be used to routinely identify acute myocardial infarctions because of other diagnostic techniques (Khaw, 1999). This agent, however, may be used to diagnose and evaluate acute myocarditis (Narula et al., 1999), cardiomyopathies (Nanas et al, 2000) and chemotherapy-induced cardiotoxicity (Carrio et al., 1993). NORMAL LOCALIZATION AND RADIATION DOSIMETRY Indium-111-MyoScint remains in the blood with an initial short T1/2 of 1.5 hr and slower phase washout of 20.2 hr (MyoScint, 1996). At 24 hr, 24% of the initial activity remains in the plasma. Even though this radiopharmaceutical is a Fab fragment, imaging must wait for ~24 hr, in order to let the blood pool radioactivity clear. The 111In activity remaining is usually localized in the liver, spleen, and kidney and at later time points, bone marrow. The kidneys receive the greatest concentration of radionuclide. MyoScint does not localize in old infarcted areas where scar has formed. Maximum antibody uptake occurs in areas of lowest perfusion. HAMA response has not been observed in patients, even with repeated injections. The dosimetry for a 74 MBq [2 mCi] dose is kidneys = 8.8 cGy, liver = 4.5, spleen = 3.4, bone marrow = 3.2, and ovaries = 0.8. INDIUM-111 LABELED DTPA (111In-DTPA) PREPARATION AND QC To prepare this radiopharmaceutical 37-74 MBq of 111InCl3 is added to solution containing 0.05 M acetate buffer pH 4 to 5 (Kowalsky & Perry, 1987b). Then 5-20 mg of DTPA is added, pH readjusted to 5 and the mixture incubated for 15-30 min in a boiling water bath (Fig. 2 and 7B). Colloidal 111In is removed, if necessary, by gel permeation chromatography. RCP is determined using Whatman Paper No. 1 and saline as the solvent. The Rf of 111In-DTPA is 0.8 and for hydrolyzed colloidal 111In is 0.0. The dose is 55.5 MBq, and the preparation is good for 24 hr.

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INDICATIONS FOR SCINTIGRAPHY This radiopharmaceutical is used to determine the distribution of cerebrospinal fluid (CSF) and more specifically whether there is a leak in the central nervous system (CNS) (Servadei et al., 1998; Iino et al., 2000; Chamberlain, 1998). This special fluid, CSF, bathes the CNS, which includes the brain and the spinal canal. Blood is prevented from entering the CNS by the blood-brain barrier. CSF is produced in the central part of the brain near the hypothalamus, the ventricles, circulates throughout the brain and spinal chord. The CSF is finally absorbed in an area at the top of the brain near the skull, the arachnoid villi. NORMAL LOCALIZATION AND RADIATION DOSIMETRY After intrathecal injection into spinal canal space, the 111In-DTPA moves with the flow of CSF down the spinal canal to the head (Kowalsky & Perry, 1987c). The nuclide migrates to the base of the brain (basal cisterns), over the top of the cerebral cortex to the parasagittal space (region between top brain and brain membrane) and finally to the arachnoid villi where it exits. The 111In-DTPA does not normally enter the ventricle space. Activity reaches the cisterns in an hour, peaking at 4 hr. Parasagittal radioactivity is first seen at 4 hr, maximizes at 14 to 17 hr, and declines with a T1/2 of 10–14 hr. The radionuclide then appears in the blood and is rapidly excreted by the kidneys. Approximately 65% of the dose is excreted in the first 24 hr and 85% in 72 hr. The absorbed dose for spinal cord averages 1.5 cGy/18.5 MBq, for brain is 0.4, and kidney is 0.22 (indium-111-DTPA, 1990). INDIUM-114M-OXINE LABELED LYMPHOCYTES (114mIn-Lymphocytes) PREPARATION AND QC Indium-114m-lymphocytes are prepared by removing ~ 50 mL of the patients blood, isolating the white cell fraction from the plasma, layering the mixed leukocytes on a density gradient material (Lymphoprep) and centrifuging this mixture at low speed (400 g for 40 min) [Sharma et al., 1997]. The band containing the lymphocytes and monocytes is carefully removed and washed free of plasma. This fraction is re-suspended in buffer and incubated for ~15 min with 114mIn-oxine to label the cells at ambient temperature. The plasma is added back to cells and the cells centrifuged to remove non-bound activity. The cells are re-suspended in plasma and finally injected into the patient. The viability of labeled cells after labeling is determined by a dye exclusion method. The therapeutic dose range was from 69-211 MBq. INDICATIONS FOR SCINTIGRAPHY Indium-114m-lymphocytes are being investigated for the treatment of refractory chronic lymphocytic leukemia. Most recently, patients have been treated for low-grade non-Hodgkin's lymphoma (Sharma et al., 1997). The large excess of lymphocytes in the blood of these patients makes it easy to isolate a large quantity of autologous cells for labeling. NORMAL LOCALIZATION After injection a large fraction of the 111In is distributed mostly in the spleen (53%), some in the liver (35%) and 5% in the bone marrow (Sharma et al., 1997).

Initially there was a rapid decline of the lymphocyte

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count in the peripheral blood. No patient had subjective toxicity but all had myelosuppression. There was a response in 10 of 14 patients. Two patients had a complete response and 8 had only a partial response. FUTURE DIRECTIONS Gallium-67 has been used as a tumor-imaging agent for over 30 years. This nuclide continues to play a major role in the diagnosis and staging of Hodgkin's disease, lymphoma and melanoma. During the past few years, however, 18F-fluorodeoxyglucose (18F-FDG) has drawn considerable attention as a metabolic tracer for imaging neoplasms by positron emission tomography (PET). This has culminated in the approval of this technique for staging and diagnosis of colorectal cancer, lymphoma and melanoma. The success of 18F-FDG has led to the commercial development of less expensive, coincidence gamma cameras and concomitantly increased supplies of radiopharmaceutical. It is possible in the future that the role of 67Ga may be diminished. However, because of large cost advantage of 67Ga as well as long T1/2 this radionuclide may continue to contribute to the management of certain cancer patients. However, in light of the potential of 99m Tc-based agents, the use of 67Ga for infections/inflammation imaging may be less certain. The increased clinical utilization of PET technology may reduce the utilization of one gallium radioisotope but it may increase the use of the other, 68Ga. The increased availability of PET and coincidence gamma cameras in large and small hospitals throughout the USA may stimulate research, both public and private, into the development of clinically useful 68Ga compounds. The great allure to such development is the potential of paralleling the success of the 99Mo/99mTc generator with a ready supply of inexpensive 68Ga. At present 18F-FDG is the only approved radiopharmaceutical for PET imaging and is available from few sites throughout the country. The short T1/2 of 18F, 110 min, could mean that a cyclotron must be within a few hours of the hospitals it serves. In contrast, the long T1/2 of 68Ge (275 d) means that this generator could supply 68Ga for over two years without being replaced. The use of 111In-WBC is also diminished by competing radiopharmaceuticals. Some of these such as 99mTcWBC are already in use and others are on the horizon. There are at least two new99mTc-based WBC-directed antibodies that are presently in clinical trials (Barron et al., 1999; Kipper et al., 2000). One of these, LeuTech is likely to receive FDA approval (Thakur personal communication). The advantage of these agents, besides the utilization of 99mTc, is that they do not require blood isolation and will likely be able to make the final diagnosis within 4 hr of injection compared to the 24 hr for 111In-WBC. This adds to the strength of these agents since rapid diagnosis reduces morbidity and mortality and costs associated with patient management. In contrast, the use of 111In for detection of tumors using peptides or antibodies has had explosive growth. Numerous investigations are in progress to develop both antibody and peptide-based radiopharmaceuticals to diagnose and treat a variety of cancers. Efforts are particularly concentrated on those cancers, such as ovarian, where present techniques are woefully inadequate (Kalofonos et al., 1999; Davies et al., 1999; de Jong et al., 1999; Wong et al., 1998; Barbet et a.l, 1998; Clarke et al., 1999). Initial studies will continue to utilize 111In because of its ability to bind with high affinity to biomolecule conjugated DTPA or other similar chelates. If the localization process requires one or more days, then 111In will be the most appropriate

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radionuclide. However, if the localization can be manipulated to reduce the time to less than one day then 99m Tc may be the radionuclide of choice. This radionuclide has much lower cost and better imaging characteristics than 111

111

In. The most recently approved tumor-detecting radiopharmaceuticals have contained

99m

both In and Tc (ProstaScint, 1996; OncoScint, 1992; CEA-Scan, 1996; NeoTect 1999; Verluma, 1996; OctreoScan, 1994). For cancer diagnosis it is not necessary to image the patient quickly after the injection. The driving force for these investigations is to develop a technique that has both high sensitivity and specificity. The objective is to detect all the cancerous lesions. This allows the patient to be staged appropriately. Also, full detection can determine if the disease has re-occurred or remained after a course of therapeutic or surgical intervention. Many new therapeutic strategies use 90Y (DeNardo et al., 2000; Wiseman et al., 2000; Cremonesi et al., 1999; Pai-Scherf et al., 2000). This nuclide has high-energy (3, maximum 2.28 MeV but no y photon for imaging. In many cases, both 111In and 90Y have high affinity to the same chelating agents and biodistributions of both chelate conjugates are similar. Thus, 111In has been used to follow the kinetics and the biodistribution of these new radiopharmaceuticals in patients. This is prior to receiving a therapeutic dose of the 90Y radiopharmaceutical. For example, 111In labeled Zevalin ( 111 In-Zevalin), an anti-B cell directed antibody, has been used to tailor the appropriate dose for the 90Y-labeled Zevalin (90Y-Zevalin) [Wiseman et al., 2000]. The 111In-Zevalin allows imaging of both the target and non-target tissue over time which would be much more difficult with 90Y-Zevalin. In this manner the absorbed dose for non-target tissue, which limits the total dose given, can be estimated more accurately. Both gallium and indium radionuclides have had a long useful history in Nuclear Medicine. While various aspects of their utilization may wax and wane, we anticipate that both will continue to play an important role in our discipline. ACKNOWLEDGEMENTS We would like to thank John Murren, MD and his group at Yale University, School of Medicine, for generously providing unpublished images of a patient undergoing therapy with 111In-OcteoScan. The assistance of Pricilla Paretta in the preparation of this manuscript is gratefully acknowledged.

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antibody (hCTMOl) for use in the treatment of ovarian carcinoma, Br. J. Obstet. Gynaecol., 106, 31– 37, deJong M, Breeman WA, Bernard HF, Kooij PP, Slooter GD, Van Eijck CHJ, Kwekkeboom DJ, Valkema R, Macke HR and Krenning EP (1999) Therapy of neuroendocrine tumors with radiolabeled somatostatin-analogues. Q. J. Nucl. Med., 43, 356–366. DeNardo GL, O'Donnell RT, Shen S, Kroger LA, Yuan A, Meares CF, Kukis DL and DeNardo SJ (2000) Radiation dosimetry for 90Y-21T-BAD-Lym~l extrapolated from pharmacokinetics using 111In-21TBAD-Lym-1 in patients with non-Hodgkin's lymphoma. J. Nucl. Med., 41, 952-958. Dudley HC and Garzoli RF (1948) Preparation and properties of gallium lactate. J. Amer. Chem. Soc., 70,

3942-3943. Dudley HC, Maddox GE and La Rue HC (1949) Studies of the metabolism of gallium. J. Pharm. Exp. Ther., 96, 135–138. Edwards CL and Hayes RL (1969) Tumor scanning with 67Ga citrate. J. Nucl. Med., 10, 103–105. Elagamal AA, Troy MJ and Murphy GP (1998) ProstaScint scan may enhance identification of prostate cancer recurrences after prostatectomy, radiation, or hormone therapy: Analysis of 136 scans of 100 patients. Prostate, 37, 261-269. Gallium citrate (1992) 67Ga injection, Package insert, Mallinckrodt, St Louis. Gardin I, Faraggi M, Le Guludec D and Bok B (1999) Cell irradiation caused by diagnostic nuclear medicine procedures: Dose heterogeneity and biological consequences. Eur. J. Nucl. Med., 26, 1617-1626. Gibril F, Reynolds JC, Chen CC, Yu F, Goebel SU, Serrano J, Doppman JL and Jensen RT (1999) Specificity of somatostatin receptor scintigraphy: A prospective study and effects of false-positive localizations on management in patients with gastrinomas. J. Nucl. Med., 40, 539-553. Goodwin DA, Finston RA, Colombetti LG, Beaver JE and Hupf H (1969) 111In-Colloid for lymphatic scintiphotography. J. Nucl. Med., 10, 337. Goodwin DA, Sundberg MW, Diamanti CI and Meares CF (1975) 111In-labeled radiopharmaceuticals and their clinical use. In Radiopharmaceuticals, Subramanian G, Rhodes BA, Cooper JF, and Sodd VJ (eds), Society of Nuclear Medicine, New York, 80–101. Green MA and Huffman JC (1988) The molecular structure of indium oxine. J. Nucl. Med., 29, 417–420. Harker LA and Finch CA (1969) Thrombokinetics in man. J. din. Inves., 48, 963-974. Harris WR, Chen YC and Wein K (1994) Equilibrium constants for the binding of indium(III) to human serum transferrin. Inorg. Chem., 33, 4991–4998. Harris WR and Pecoraro VL (1983) Thermodynamic binding constants for gallium transferrin. Biochemistry, 22, 292-299. Hayes RL (1978) The medical use of gallium radionuclides: A brief history with some comments. Sem. Nuc. Med., 8, 183-191. Hnatowich DJ (1990) Antibody radiolabeling, problems and promises, Nuc. Med. Biol., 17, 49-55. Hunter Jr. WW and DeKock HW (1969) 111In for tumor localization. J. Nucl. Med,. 10, 343. Iino K. Yoshinari M, Yoshizumi H, Ichikawa K, Iwase M and Fujishima M (2000) Normal pressure hydrocephalus in diabetic patients with recurrent episodes of hypoglycemic coma. Diabetes Res. Clin. Pract., 47, 105–110. Indium Chloride (1995) sterile solution, Package insert, Mallinckrodt, St Louis.

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Indium-111 Oxine Solution (1986) Package insert, Amersham Healthcare, Arlington Heights. Indium-111 DTPA (1990) Package insert, Amersham Healthcare, Arlington Heights. Jackson GE and Byrne MJ (1996) Metal ion speciation in blood plasma: Gallium-67-citrate and MRI contrast agents. J. Nucl. Med,, 37, 379-386. Kalofonos HP, Giannakenas C, Kosmas C, Apostolopoulos D, Onienadum A, Petsas T, Dimopoulos D. Epenetos AA and Vassilakos PJ (1999) Radioimmunoscintigraphy in patients with ovarian cancer. Acta. Oncol., 38, 629-634. Kaplan WD (1990) Editorial: Residual mass and negative gallium scintigraphy in treated lymphoma: When is the gallium scan really negative. J. Nucl. Med., 31, 369. Khaw BA (1999) The current role of infarct avid imaging. Semin. Nucl. Med., 29, 259-270. Kipper, SL, Rypins EB, Evans DG, Thakur ML, Smith TD and Rhodes B (2000) Neutrophil specific 99mTclabeled anti-CD15 monoclonal antibody imaging for diagnosis of equivocal appendicitis. J. Nucl. Med., 41, 449-455. Kowalsky RJ and Perry JR (1987a) Radiopharmaceuticals in Nuclear Medicine Practice. Appleton & Lange, Norwalk, 379-387. Kowalsky RJ and Perry JR (1987b) Radiopharmaceuticals in Nuclear Medicine Practice. Appleton & Lange, Norwalk, 388-406. Kowalsky RJ and Perry JR (1987c) Radiopharmaceuticals in Nuclear Medicine Practice. Appelton & Lange, Norwalk, 157–176. Krenning EP, Kwekkeboom DJ, Bakker WH, Breeman WAP, Kooij PPM, Oei HY, van Hagen M, Posterma PTE, de Jong M, Reubi JC, Visser TJ, Reijs AEM, Hofland LJ, Koper JW and Lamberts SWJ (1993) Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe1] and [123I-Tyr3]-octreotide: The Rotterdam experience with more than 1000 patients. Eur. J. Nucl. Med., 20, 716–731. Krenning EP, de Jong M, Kooip PP, Breeman WA, Bakker WH, de Herder WW, van Eijck CH, Kwekkeboom DJ, Famar F, Pauwels S and Walkema R (1999) Radiolabelled somatostatin analogue(s) for peptide receptor scintigraphy and radionuclide therapy. Ann. Oncol., 10, S23-S29 Lamb JF (1982) Commercial production of isotopes for nuclear medicine, 1970–1980. IEEE Trans. Nuc. Sci., NS-28, 1916–1920. Larson, SM, Rasey JS, Allen DR, Nelson NJ, Grunbaum Z, Harp GD, and Williams DL (1980) Common pathway for tumor cell uptake of gallium-67 and iron-59 via a transferrin receptor. J. Natl. Can. Inst., 64, 41–53 Latimer WM (1952) The Oxidation States of the Elements and Their Potentials in Aqueous Solution, 2nd edition, Prentice-Hall, New York, 158–167. Lavender JP, Lowe J and Barker JR. (1971) Gallium-67 citrate scanning in neoplastic and inflammatory lesions. Br. J. Radiol., 44, 361–366. Lederer CM and Shirley VS (eds) (1978a) Table of Isotopes, 7th ed, John Wiley & Sons. Inc.. New York. 207-216. Lederer CM and Shirley VS (eds) (1978b) Table of Isotopes, 7th ed, John Wiley & Sons, Inc.. New York. 508-536. Loc'h C, Maziere B and Comar D (1980) A new generator for ionic gallium-68.7. Nucl. Med., 21. 171–173.

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Logan KJ, Ng PK, Turner CJ, Schmidt RP, Terner UK, Scott JR, Lentle BC and Noujaim AA (1981) Comparative pharmacokinetics of 67Ga and 59Fe in humans. Int. J. Nucl. Med. Biol., 8, 271–276. Louwes H, Zeinali Lathori OA, Vellenga E and de Wolf JT (1999) Platelet kinetic studies in patients with idiopathic thrombocytopenic purpura. Am. J. Med., 106, 430-434. Macke HR, Riesen A and Ritter W (1989) The molecular structure of indium-DTPA. J. Nucl. Med., 30, 1235-1239. Manyak MJ, Hinkel GH, Olsen JO, Chiaccherini RP and Partin AW (1999) Immunoscintigraphy with indium-111-capromab pendetide: Evaluation before definitive therapy in patients with prostate cancer. Urology, 54, 1058-1063. Martell AE and Hancock RD (1996) Metal Complexes in Aqueous Solutions. Plenum Press, New York, pp. 166-171. McAfee JG and Thakur ML (1975) Survey of radioactive agents for in vitro labeling of phagocytic leukocytes. I. soluble agents. J. Nucl, Med., 17, 488–493. McCarthy KE, Woltering EA and Anthony LB (2000) In situ radiotherapy with 111In-pentetreotide. State of the art and perspectives. Q. J. Nucl. Med., 44, 88-95. McElvaney KD, Hopkins KT, Hanrahan TJ, Moore HA and Welch MJ (1982) Comparison of Ge-68/Ga-68 generator system for radiopharmaceutical production. J. Labelled Compd. Radiopharm., 19, 1419– 1420. McElvaney KD, Hopkins KT and Welch MJ (1984) Comparison of germanium-68 generator systems for radiopharmaceutical production, Int. J. Appl. Radiat. Isot., 35, 521-524. Murphy GP, Elgamal AA, Troychak MJ and Kenny GM (2000) Follow-up ProstaScint scans verify detection of occult soft-tissue recurrence after failure of primary prostate cancer therapy. Prostate 42, 315–317. MyoScint (1996) Package insert, Centocor, Malvern. Nanas JN, Margari ZJ, Lekakis JP, Alexopoulos GE, Prassopoulos V, Agapitos EV. Toumanidis ST, Anastasiou-Nana MI, Kostamis P and Stamatelopoulos SF (2000) Indium-111 monoclonal antimyosin cardiac scintigraphy in men with idiopathic dilated cardiomyopathy. Am. J. Cardiol., 85, 214–220. Narula J, Malhotra A, Yasuda T, Talwar KK, Reddy KS, Chopra P, Southern JF, Vasan RS, Tandon R, Bhatia ML, Khaw BA and Strauss HW (1999) Usefulness of antimyosin antibody imaging for the detection of active rheumatic myocarditis. Am. J. Cardiol., 84, 946-950. Nelson B, Hayes RL, Edwards CL, Kniseley RM and Andrews GA (1972) Distribution of gallium in human tissues after intravenous administration. J. Nucl. Med., 13, 92–100. NeoTect (1999) Package insert, Diatide, Londonderry. Neumann RD, Kemp JD and Weiner RE (1995) Gallium-67 imaging for detection of malignant disease. In Diagnostic Nuclear Medicine, Sandier MP, Coleman RE, Wackers FJTh, Patton JA, Gottschalk A, HofferPB (eds), 3rd ed., Williams & Wilkins, Baltimore, 1243-1260. Neumann RD, and McAfee (1995) Gallium-67 imaging in infection. In Diagnostic Nuclear Medicine, Sandier MP, Coleman RE, Wackers FJTh, Patton JA, Gottschalk A, Hoffer PB (eds), 3rd ed., Williams & Wilkins, Baltimore, 1493–1507. OctreoScan (1994) Package insert, Mallinckrodt, St Louis, MO. OncoScint (1992) Package insert, Cytogen, Princeton, N.J.

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Otte A, Jermann E, Behe M, Goetze M, Bucher HC, Roser HW, Heppeler A, Mueller-Brand J, and Macke HR (1997) DOTATOC: A powerful new tool for receptor-mediated radionuclide therapy. Eur. J. Nucl. Med., 24, 792–795. Pai-Scherf LH, Carrasquillo JA, Paik C, Gansow O, Whatley M, Pearson D, Webber K, Hamilton M, Allegra C, Brechbiel M, Willingham MC and Pastan I (2000) Imaging and phase I study of 111In- and 90Ylabeled anti-LewisY monoclonal antibody B3. Clin. Cancer Res., 6, 1720–1730. Pecoraro VL, Wong GB and Raymond KN (1982) Gallium and indium imaging agents. 2. Complexes of sulfonated catecholamine sequestering agents. Inorg. Chem., 21, 2209-2215. Pinkas L, Robins PD, Forstrom LA, Mahoney DW and Mullan BP (1999) Clinical experience with radiolabelled monoclonal antibodies in the detection of colorectal and ovarian carcinoma recurrence and review of the literature. Nucl. Med. Commun., 20, 689-696. Polascik TJ, Manyak MJ, Haseman MK, Gurganus RT, Rogers B, Maguire RT and Partin AW (1999) Comparison of clinical staging algorithms and 111indium-capromab pendetide immunoscintigraphy in the prediction of lymph node involvement in high risk prostate carcinoma patients. Cancer, 85, 15861592. Preston DF (1995) Indium-111 label in inflammation and neoplasm imaging. In Principles and Practice of Nuclear Medicine. Early PJ and Sodee DB(eds), Mosby, St Louis, 714–724. ProstaScint (19%) Package insert, Cytogen, Princeton, N.J. Quintana JC and Blend MJ (2000) The dual-isotope ProstaScint imaging procedure: Clinical experience and staging results in 145 patients. Clin. Nucl. Med., 25, 33–40. Reichart DE, Lewis JS and Anderson CJ (1999) Metal complexes as diagnostic tools. Coord. Chem. Rev., 184, 3–66. Rodriques M, Sinzinger H, Thakur M, Becker W, Dewanjee M, Ezekowitz M, Isaka Y, Martin-Comin J, Peters M, Roca M and Stratton J (1999) Labelling of platelets with indium-111 oxine and technetium99m hexamethylpropylene amine oxine: Suggested methods. International Society of Radiolabelled Blood Elements (ISORBE). Eur. J. Nucl. Med., 26, 1614–1616. Scheffel U, Tsan M-F, Mitchell TG, Camargo EE, Braine H, Ezekowitz D, Nickoloff EL, Hill-Zobel R, Murphy E and McIntyre PA (1982) Human platelets labeled with In-111 8-hydroxyquinoline: Kinetics, distribution, and estimates of radiation dose. J. Nucl. Med., 23, 149–156. Servadei F, Moscatelli G, Giuliani G, Cremonini AM, Piazza G, Agostini M and Riva P (1998) Cisternography in combination with single photon emission tomography for the detection of the leakage site in patients with cerebrospinal fluid rhinorrhea: preliminary report. J. Nucl. Cardiol., 5. 551-557. Sharma HI, Cowan RA, Murby B, Owens S, Nuttall PM, Gunton D, Smith AM, Chang J and Crowther D (1997) Treatment of lymphoid cell malignancies with 114In-labelled autologous lymphocytes. Nucl. Med. Commun., 18, 474. Silvester DJ (1978) Consequences of indium-111 decay in vivo: Calculated absorbed radiation dose to cells labeled by indium-111 oxine J. Labelled Comp. Radiopharm., 19, 196-197. Thakur ML, Coleman RE and Welch MJ (1977a) Indium-111-labeled leukocytes for the localization of abscesses: Preparation, analysis, tissue distribution, and comparison with gallium-67 citrate in dogs. J. Lab. Clin. Med., 89, 217–228.

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Thakur, ML, Segal AW, Louis L, Welch MJ, Hopkins J and Peters TJ (1977b) Indium-111-labeled cellular blood components: Mechanism of labeling and intracellular location in human neutrophils, J. Nucl. Med., 18, 1022–1027. Thakur ML, Lavender JP, Amot RN, Silvester DJ and Segal AW (1977c) Indium-111-labeled autologous leukocytes in man. J. Nucl. Med., 18, 1014–1021. Thakur ML (1977) Gallium-67 and Indium-111 Radiopharmaceuticals. Int. J. App. Rad. Isot., 28, 183–201. Thompson M (1978) A study of 8-hydroxyquinolinates of some trivalent metal ions by potentiometry and xray photoelectron spectroscopy. Anal. Chim. Acta., 98, 357-363. Tiensuu Janson E, Westlin J-E, Ohrvall U, Oberg K and Lukinius A (2000) Nuclear localization of 111In after intravenous injection of [111In-DTPA-D-Phe1]-octreotide in patients with neuroendocrine tumors. J. Nucl. Med., 41, 1514-1518. Tolmachev V, Bernhardt P, Forssell-Aronsson E and Lundqvist H (2000) 114mIn, a candidate for radionuclide therapy: Low-energy cyclotron production and labeling of DTPA-D-phe-octreotide. Nucl. Med. Biol., 27, 183–188. van Eijck CH, de Jong M, Breeman WA, Slooter GD, Marquet RL and Krenning EP (1999) Somatostatin receptor imaging and therapy of pancreatic endocrine tumors. Ann. Oncol., 10 1777–1781. Verluma (1996) Package insert, DuPont Pharma, Billerica, MA Weiner RE, Spencer RP, Dambro TJ and Klein BE (1992) Gallium-67 distribution in a man with both a decrease in transferrin and hepatic gallium-67 concentration. J. Nucl. Med., 33, 1701–1703. Weiner RE (1996) The mechanism of 67Ga localization in malignant disease. Nucl. Med. Biol., 23, 745–751. Weiner RE and Thakur ML (1995) Metallic Radionuclides: Applications in diagnostic and therapeutic nuclear medicine, Radiochim. Acta., 70/71, 273–287. Welch MJ and Welch TJ (1975) Solution chemistry of carrier free indium. In Radiopharmaceuticals, Subramanian G, Rhodes BA, Cooper JF and Sodd VJ (eds), Society of Nuclear Medicine, New York, 73-79. Wiseman GA, White CA, Stabin M, Dunn WL, Erwin W, Dahlbom M, Raubitschek A, Karvelis K, Schultheiss T, Witzig TE, Belanger R, Spies S, Silverman DH, Berlfein JR, Ding E and Grillo-Lopez AJ (2000) Phase I/II 99Y-Zevalin (yttrium-90 ibritumomab tiuxetan, IDEC-Y2B8) radioimmunotherapy dosimetry results in relapsed or refractory non-Hodgkin's lymphoma. Eur. J. Nucl. Med., 27, 766-777. Wong JY, Chu DZ, Yamauchi D, Odom-Maryon TL, Williams LE, Liu A, Esteban JM, Wu AM, Primus FJ, Beatty JD, Shively JE and Raubitschek AA (1998) Dose escalation trial of indium-111-labeled anticarcinoembryonic antigen chimeric monoclonal antibody (chimeric T84.66) in presurgical colorectal cancer patients. J. Nucl. Med., 39, 2097-2104. Zweit J, Sharma H and Downey S (1987) Production of gallium-66, a short-lived, positron emitting radionuclide. Appl. Radiat. Isot., 38, 499-501.

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12. CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS CAROLYN J. ANDERSONA, MARK A. GREENB AND YASUHISA FUJIBAYASHIC A

Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway, Campus Box 8225, St. Louis, MO 63110, U.S.A.;BDepartment of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907; U.S.A.; andC Biomedical Imaging Research Center, Fukui Medical University, 233, Shimoaizuki, Matsuoka, Yoshida, Fukui, 910-1193, Japan INTRODUCTION Copper offers a relatively large number of radioactive isotopes that are potentially suitable for use in diagnostic imaging and/or targeted radiotherapy, specifically: 67Cu, 64Cu, 62Cu, 61Cu, and 60Cu (Table 1). These copper radioisotopes present relatively diverse nuclear properties, including half-lives ranging from 10 minutes to 62 hours, and decay by positron (fT) and/or beta-minus (P>emission. Additionally, the regional tissue distribution of all of these copper radioisotopes can be externally assessed with clinical gamma or positron imaging techniques. The two longer-lived copper radionuclides, 64Cu and 67Cu, have a long history of application as biomedical tracers, notably in the use of 64Cu2+ and 67Cu2+ to probe copper absorption, metabolism and excretion (Owen, 1982), as well as for assessment of patients with Wilson's disease (Owen, 1981). They were also the subjects of early investigations in tumor imaging, either as porphyrin (Bases et al., 1963) or citrate (Raynaud et al., 1973) complexes. This review will focus on recent developments in copper radiopharmaceutical chemistry that have significantly expanded the possible clinical role of copper radioisotopes. Current research in copper radiopharmaceutical chemistry has been stimulated by: (i) the array of nuclear properties presented by copper radioisotopes; (ii) the relatively diverse coordination chemistry of Cu that can be exploited in radiopharmaceutical design; (iii) the evolution of practical production techniques for these radionuclides; (iv) the growing interest, and technical feasibility, of targeted radiotherapy with short-tomedium half-life ^'-emitting nuclides; and (v) the growing availability of clinical equipment for imaging by positron emission tomography (PET). PET is a medical imaging technique that can quantitatively map radiopharmaceutical concentrations inside the living body (Ter-Pogossian et al., 1980), thereby allowing non-invasive assessment of various aspects of tissue biochemistry and physiology through direct observation of regional drug distribution and/or pharmacokinetics. A key attribute of PET is the capability of absolute quantification of regional radionuclide distribution, avoiding problems with tissue attenuation of emitted photons that intrinsically hamper in vivo quantification of single-photon gamma-emitting nuclides. However, imaging with PET requires use of

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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HANDBOOK OF RADIOPHARMACEUTICALS

radionuclides that decay with emission of a positron (most commonly, cyclotron-produced 15O, I3 N. 11C, and 18 F), as well as imaging hardware that has only recently begun to have widespread clinical availability. Positron-emitting copper radionuclides will not replace the extremely versatile 15O, 13N, 11C, and 18F radiolabels widely employed in current PET radiopharmaceuticals. However, the positron-emitting copper nuclides do offer attractive nuclear properties for some applications of PET, along with the ability to exploit unique aspects of copper chemistry in radiopharmaceutical design. Thus, 64Cu, 61Cu, 62Cu and/or 60Cu radiopharmaceuticals may have clinical impact in roles that complement PET radiotracers now in clinical use. PRODUCTION OF COPPER RADIONUCLIDES The radionuclides of copper offer a selection of diagnostic (60Cu, 6l Cu, 62Cu and 64Cu) and therapeutic (64Cu and 67Cu) isotopes (Table 1). The positron-emitting diagnostic nuclides have a wide range of half-life (10 min to 12.7 h) and are reactor, cyclotron, or generator produced. Copper-67 is a therapeutic radionuclide that is currently produced on a high-energy accelerator. Copper-64 can be effectively produced by both reactor-based and accelerator-based methods. One method of Cu production is the 64Zn(n,p)64Cu reaction in a nuclear reactor (Zinn et al., 1994). By utilizing fast neutrons, high specific activity 64Cu was produced at the Missouri University Research Reactor (MURR) in amounts averaging 250 mCi. Smith and coworkers separated large amounts of 64Cu by-product from cyclotron production of 67Ga via the 68Zn(p,2n)67Ga reaction (Smith et al., 1996). This mode of production has the advantage of being very economical and allows for production of very large amounts (> 3 Ci) of reasonably high specific activity material (~860 Ci/mmol). The disadvantage is that on-demand production would be problematic, since the major radionuclide produced is longer-lived 67Ga (T1/2 = 72 h). More recently, large amounts of 64Cu have been produced by the 64Ni (p,n) 64Cu nuclear reaction using a biomedical cyclotron (McCarthy et al., 1997). A target has been specifically designed for the production of this radionuclide using enriched 64Ni, which can be efficiently recovered in target processing (McCarthy et al., 1997). Advantages of this method of production are that very high specific activity 64Cu is produced (>10,000 Ci/mmol), and Ci amounts can be prepared on demand. 64

Table 1. Decay Characteristics of Copper Radionuclides Isotope 60 Cu

11 T*l/2

23.4 min

| 3" MeV (%) | 3 + MeV(%) 3.92 (6%) 3.00(18%) 2.00 (69%)

EC(fc) 7.4%

61

Cu

3.32 h

-

1 .22 (60%)

40%

62

Cu Cu

9.76 min 12.8 h

0.573 (39.6%)

2.91 (97%) 0.655(19.3%)

2% 41%

Cu

62.0 h

0.577 (20%) 0.484 (35%) 0.395 (45%)

-

64 67

7MeV(%) 0.85(15%) 1.33(80%) 1.76(52%) 2.13(6%) 0.284(12%) 0.38 (3%) 0.511(120%) 0.511 (194%) 1 .35 (0.6%) 0.511(38.6) 0.184(40%) 0.092(23%)

CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS

403

By altering the enriched isotope of nickel used as a target, large quantities of 60Cu and 61Cu have also been produced on a biomedical cyclotron (McCarthy et al., 1999). Yields up to 865 mCi of 60Cu, and 144 mCi of 61 Cu, have been achieved using this method. These radionuclides have shorter half-lives (23 min and 3.3 h, respectively), and have clinical applications in PET imaging (Dehdashti et al., 2000). Copper-62 is obtained from the decay of cyclotron-produced 62Zn (T1/2 = 9.2 h). Use of the 62Zn/62Cu parent/daughter generator as a source of 62Cu for radiopharmaceuticals was first suggested by Robinson et al. (Robinson et al., 1980). Although this generator only has a limited life span of 1-2 days, there has been significant interest in 62Cu radiopharmaceuticals. Several different modes of generator production of 62Cu have been reported. The Robinson generator consists of 62Zn loaded onto a Dowex 1 x 10 anion exchange column, and the generator was eluted with 0.1 N HC1 with 100 mg/mL NaCl with 1 |ig/mL CuQ2. The carrier copper is not required for efficient separation of 62Cu from 62Zn, and Robinson-type no-carrier-added 62 Cu generators have been used extensively in assessment of 62Cu-PTSM as a PET perfusion tracer (Green, et al,, 1990; Mathias, et al., 1991; Lacy, et al, 1995; Lacy et al., 1996; Wallhaus, et al., 1998; Haynes, et al., 2000). Fujibayashi et al. loaded 62Zn in 2 mL water at pH 5.0 with < 2 jig/mL of Cu carrier on CG-120 Amberlite cation exchange resin (Fujibayashi et al., 1989). The generator eluent was 200 mM glycine. The Cu carrier was eluted from the column during the first elution. Zweit et al. developed a 62Zn/62Cu generator where the 62Zn was loaded onto a AG1-X8 column which was eluted with 0.3 M HCl/40% ethanol to give >90% 62Cu yield in 3 mL with extremely low 62Zn breakthrough of <3x 10-7 % (Zweit et al., 1992). Bormans et al. developed a 62Zn/62Cu generator based on a small (25mm x 5mm) ion exchange column (Dowex 1 x 16) that is eluted with 1.7 M NaCl and 0.1 M HC1 (Bormans et al., 1992). A commercially available 62 Zn/62Cu generator has been used for Phase III clinical trials (Haynes et al., 2000). Copper~67 is produced by a high-energy accelerator by bombarding a natural Zn target with 200 MeV protons (68Zn(p, 2p)) (Mirzadeh et al., 1986). Separation of the 67Cu from the Zn target has been accomplished by electrodeposition (Mirzadeh et al., 1986), and solvent extraction (Dasgupta et al., 1991). Production by this method also produces a large amount of 64Cu. The major disadvantage of this mode of production is that there are only two such high-energy, high-current, accelerators in the U.S., and therefore, the cost is high and the supply is limited. More recently, the production of 67Cu on low and medium-energy cyclotrons has been reported (Jamriska et al., 1995; Kastleiner et al., 1999). Kastleiner and colleagues predict that up to 400 mCi could be produced at saturation using a small cyclotron (Ep = 17-18 MeV; 80 juA beam current) (Kastleiner et al., 1999). This method of production would allow more facilities throughout the world to produce 67Cu for possible clinical use in radiotherapy and/or gamma scintigraphy. COORDINATION CHEMISTRY OF COPPER RADIOPHARMACEUTICALS CHEMISTRY OF COPPER The chemistry of copper in aqueous solution is restricted to two principal oxidation states (I and II). Copper(I) generally only exists in aqueous solution as a strong complex, since the free ion disproportionates

404

HANDBOOK OF RADIOPHARMACEUTICALS

to Cu2+ and copper metal (Cu°). The Cu3+ ion may be formed under certain conditions, but it is a powerful oxidant and is not a stable species in biochemical systems (Levitzki & Anbar, 1968). The Cu2+ ion has (f configuration, and therefore the mononuclear complexes are paramagnetic. Copper(II) prefers a coordination number of 4, with the ligands generally arranged in a square planar manner. In many cases two additional ligands may weakly bind forming an irregular, elongated octahedral configuration due to Jahn-Teller distortion. Copper(II) complexes are generally colored, with the \max and extinction coefficients dependent on the nature of the surrounding ligands. Copper(II) generally prefers nitrogen (amine, Schiff base, pyridine) and sulfur (thiosemicarbazone) ligands. Copper(I), with 10 d electrons, prefers tetrahedral coordination. Copper(I) complexes are colorless and diamagnetic, and donor groups are generally "soft", polarizable groups such as thioether, phosphine. or isonitrile.

DESIGN OF COPPER CHELATORS FOR RADIOPHARMACEUTICAL CHEMISTRY The design of copper chelators for diagnostic imaging agents has been dependent on the desired characteristics of the targeting molecule. For example, ligands that form lipophilic, neutral, copper complexes have been evaluated as blood flow agents. These copper complexes labeled with shorter-lived copper radionuclides were designed to be sufficiently stable to clear the blood, while passively and efficiently diffusing into tissues of interest, such as the heart, brain, kidney or tumor. Upon reaching the tissue of interest, complexes that release the copper radionuclide are advantageous, since the copper can then be trapped in the tissue without washout. Some examples of these complexes include the Cu(II) thiosemicarbazones first designed by Petering as anti-cancer agents (Petering, et al., 1964: VanGiessen. et al., 1968; VanGiessen, et al., 1973; Petering, 1972) and then evaluated as radiopharmaceuticals by Green (Green, 1987). Thiosemicarbazones labeled with shorter-lived copper radionuclides have also been developed to image hypoxia in the heart (Fujibayashi et al., 1999a; Fujibayashi et al., 1997) and in tumors (Lewis et al., 1999b). These agents will be discussed in detail below. The second class of copper radiopharmaceuticals are agents that may take a longer time to clear from the blood, and that are bound or transported by specific molecular targets. Copper-labeled biomolecules. such as monoclonal antibodies (mAbs) and peptides for tumor imaging, fall into this category. In order for this class of radiopharmaceutical to be effective, the copper must be stably bound to the biological molecule, requiring a chelator with high in vivo stability. For Cu(II) complexes to be stable in vivo, it has been demonstrated that kinetic inertness is more important than thermodynamic stability. Moi and co-workers (Moi et al.. 1985) showed that Cu(II) complexes of bifunctional chelators (BFCs) of EDTA and DTPA (Figure 1) rapidly dissociated in human serum and Cu(II) bound to albumin, even though the Cu(II) complexes had high thermodynamic stability constants (log K CU -EDTA = 18.7; log K Cu-DTPA = 21.4). Cu(II) has been found to have much greater kinetic inertness (and consequently in vivo stability) with macrocyclic chelators than with linear polyamino-polycarboxylate ligands (Cole et al., 1986). Kukis and co-workers (Kukis et al., 1994) showed that there is differential biological stability between various macrocyclic chelators. with Cu-labeled DOTA BFCs beine more stable in serum than Cu-labeled TETA BFCs. Jones-Wilson and colleagues (Jones-Wilson

CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS

405

et al., 1998) compared the thermodynamic stability of six Cu(II) macrocyclic chelator complexes, differing in carbon backbone and charge, with their in vivo behavior, and confirmed the trends shown by the research of the Meares' group (Figure 1). In addition, it was shown that the charge of the Cu(II) complex was very important in the biodistribution, and negatively-charged complexes cleared through the body much more quickly than did positively-charged agents.

V~\

CA A" H

N_

I

H

cyclen

D02A

DOTA

HOOC

^

/N

HOOC.

cyclam

et-cyclam

,

COOH

,COOH|

TETA

Figure 1. Structures of macrocyclic ligands that have been evaluated for stably complexing copper radionuclides in vivo

IV. BIOCHEMISTRY OF COPPER Copper is an essential trace metal in biological systems, often behaving as a biochemically significant catalyst (O'Dell, 1976). A large number of Cu-containing enzymes have been isolated and most contribute to redox reactions (Table 2). Additionally, a series of Cu-containing proteins, as well as small molecular-size copper complexes, have a wide range of redox potentials, and it is thought that an appropriate selection of ligands allows us to use Cu-complexes as mimics of biologically active Cu-proteins. For example, various Cu-complexes have been proposed as superoxide dismutase (SOD) mimics (Wada et al., 1994a; Wada et al., 1994c),

HANDBOOK OF RADIOPHARMACEUTICALS

406 Table 2. Cu-containing enzymes in biological system.

Protein or Enzyme

Source

Function

Cytochrome Oxidase

Mitochondria

Reduction of oxygen

Superoxide dismutase

Cytosol

2O2 to H2O2 + O2

Dopamine hydroxylase

Adrenal

Oxidation of Dopamine to Noradrenaline

Ceruloplasmin

Plasma

Oxidation of Fe(II) to Fe(III) Iron transportation

Amine oxidase

Removal of transmitters (e.g. Adrenaline)

Lysine oxidase

Aorta

Oxidation of collagen

Nitrous oxide reductase

Bacteria

Reduction of NO to N2

Tyrosinase

Skin

Oxidation of tyrosine to melanine

Based

on

the

possible

affinity

of

Cu-complexes

to

bioreductive

reaction,

anticancer

Cu-

bis(thiosemicarbazone) (Cu-BTS) complexes have been proposed (Mailer & Petering, 1976; Minkel & Petering, 1978; Minkel et al., 1978; Petering, 1977). Unfortunately, these were not clinically applicable as therapeutics, and interest gradually moved to tissue accumulation of radioactive Cu-BTS complexes in tumors (Pastakia et al., 1980). This initiated the study of biologically interesting Cu-BTS complexes as radiopharmaceuticals. Cu-PTSM (Figure 2) is the first clinically applicable Cu-BTS complex with quantitative retention into various tissues, such as brain (Green et al., 1990; Mathias et al., 1990; Okazawa et al., 1995; Okazawa et al., 1996a; Okazawa et al., 1994; Okazawa et al., 1996b), heart (Beanlands et al., 1992; Green, 1987; Tadamura et al., 1996; Wada et al., 1994b), kidneys and tumors (Mathias et al., 1994). At first, reductive retention of Cu-PTSM was thought to be a simple chemical reaction with biological reductants, such as ubiquitous intracellular glutathione (Figure 3). However, further biochemical analysis indicated that Cu-PTSM can also be reduced by a mitochondrial electron transport enzyme, in particular, Complex I in a NADH (nicotinamide adenine dinucleotide hydride)-dependent manner (Fujibayashi et al., 1995; Fujibayashi et al., 1993; Taniuchi et al., 1995). Cu-PTSM has been considered as a blood flow agent, because it is retained almost quantitatively in most tissues following relatively high first-pass extraction. However, Cu-PTSM showed hyperfixation in the brain of ischemia-reperfusion injury after transient ischemia-reperfusion (Taniuchi et al., 1997). In such tissues, disturbed electron flow was found and abnormally high electron concentration was considered to be a cause of the hyperfixation. At this point, it was clear that Cu-BTS complexes could be evaluated as radiopharmaceuticals having affinity to redox enzyme(s) as substrate mimics. This finding induced the next phase of Cu-BTS radiopharmaceutical for hypoxia imaging, described in a later section. The essential nature of Cu in biological systems has been acknowledged for a long time, but the clinical

CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS

407

application of Cu-complexes as mimics has not been pursued until more recently. The application of copper complexes as radiopharmaceuticals is one of the first examples of bridging the biochemistry of Cu with clinical applications. V. COPPER-LABELED BLOOD FLOW AGENTS Studies of myocardial and cerebral perfusion (i.e., blood flow at the capillary level) represent important clinical applications of nuclear medicine, due to the high rates of morbidity and mortality associated with cardiovascular and cerebrovascular disease. PET could improve diagnostic accuracy in these applications, compared to current nuclear medicine procedures that instead routinely employ single-photon-emitting radiopharmaceuticals, leading to interest in development of radiopharmaceuticals for myocardial and/or cerebral perfusion imaging using generator-produced

62

Cu.

Copper(II) BTS complexes (Figure 2) have been rather extensively evaluated for use as

62

Cu-

radiopharmaceuticals for perfusion imaging with PET (Beanlands et al., 1992; Bormans et al., 1992; Green, 1996; Green et al., 1990; Haynes et al., 2000; Herrero et al., 1996; Herrero et al., 1993; John & Green, 1990; Lee et al., 1991; Mathias et al., 1994; Mathias et al., 1990; Melon et al., 1994; Okazawa et al., 1994; Shelton et al., 1989; Shelton et al., 1990; Tadamura et al., 1996; Wallhaus et al., 1998; Zweit et al., 1992). These uncharged, lipophilic 62Cu-agents are attractive for clinical use because they exhibit high first-pass tissue extraction of tracer (Mathias et al., 1990; Shelton et al., 1989), insuring a good correlation between regional concentration of the radiolabel and the regional rate of tissue perfusion. In addition, the high tissue extraction of 62Cu following intravenous 62Cu-BTS administration is accompanied by prolonged tissue retention of the 62Cu-radiolabel (Figure 3). Such agents are especially appealing for use in PET perfusion studies because "long" (10-15 minute) image acquisition periods can be employed to improve counting statistics in images reconstructed at high spatial resolution. Consequently, the 62Cu-BTS complexes have shown good promise for clinical use in PET measurements of tissue blood flow, ideally exploiting the 10 minute 62Cu half-life and offering hospitals the ability to rely on a relatively inexpensive generator system for radionuclide production.

NHR,

NHR

NHR,

NHR

Figure 2. Synthesis and structural formula of the copper(II) bis(thiosemicarbazone) complexes (Cu-PTSM; R1 = R2 R3 = -CH3; Cu-ETS: R1 = -CH2CH3, R2 = R3 = H; Cu-n-PrTS: R1 = -CH2CH2CH3, R2 = R3 = H).

The lead compound in this series, Cu-PTSM, exhibited significant potential as a multi-organ tracer for flow quantification (mL-min-1•g-1) in a variety of animal models (John & Green, 1990; Mathias et al., 1994;

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HANDBOOK OF RADIOPHARMACEUTICALS

Mathias et al., 1990; Shelton et al., 1989; Shelton et al., 1990). Human PET studies with 62Cu-PTSM (Beanlands et al., 1992; Green et al., 1990; Haynes et al., 2000; Herrero et al., 1996; Herrero et al., 1993; Melon et al., 1994; Tadamura et al., 1996; Wallhaus et al., 1998) have shown this tracer to provide high quality images of the heart at rest that qualitatively and quantitatively map the pattern of myocardial perfusion. In addition, 62Cu-PTSM is a sufficiently sensitive tracer of cerebral blood flow to allow mapping of focal cerebral blood flow elevations that occur in response to neurological stimulation (Green et al., 1990; Lee et al., 1991).

62

Cu-PTSM has also been used in detection of regional cerebral perfusion impairment in

stroke patients (Okazawa et al., 1994). Cell Membrane

Cu"-L

Cu"-L

^— ^

2-

-*- HoL

Cu2+- • • • Protein

Figure 3. Schematic diagram illustrating the known thiol-mediated intracellular decomposition of the Cu11bis(thiosemicarbazone) complexes (Cu-L) (Minkel et al., 1978; Winkelmann et al., 1974). This process is believed to account, at least in part, for the prolonged tissue retention of the 62Cu-radiolabel that occurs following intravenous administration of 62Cu-PTSM and related agents (John & Green, 1990; Mathias et al., 1990). The uncharged lipophilic copper(II) bis(thiosemicarbazone) complexes readily diffuse across cell membranes, whereupon they are susceptible to reductive decomposition by reaction with ubiquitous intracellular thiols, such as glutathione (Meister & Anderson, 1983; Minkel et al., 1978; Winkelmann et al., 1974). Electron transfer to the CuII-bis(thiosemicarbazone) complex leads to dissociation of Cu(I) from the bis(thiosemicarbazone) ligand, whereupon the radiocopper ion can become bound (and effectively trapped) by intracellular macromolecules (Baerga et al., 1992; Minkel et al., 1978; Winkelmann et al., 1974). Such agents are also susceptible to reduction in mitochondria (Fujibayashi et al., 1995; Fujibayashi et al., 1993: Taniuchi et al., 1995).

However, in humans the myocardial uptake of 62Cu-PTSM is markedly attenuated at high rates of flow (contrary to experience in the dog model (Herrero et al., 1993)), seriously undermining the tracer's potential clinical utility for quantification of myocardial perfusion under hyperemic conditions (Beanlands et al., 1992; Herrero et al., 1996; Melon et al., 1994; Tadamura et al., 1996). Subsequent work (Mathias et al., 1993: Mathias et al., 1995) has revealed the source of this problem to be a substantial interspecies variability in the binding of Cu-PTSM to serum albumin (i.e., Khuman albumin > Kdog albumin): Cu-PTSM + Albumin

(Cu-PTSM)

Albumin

K=

[(Cu-PTSM)

Albumin]

[Cu-PTSM] [Albumin]

CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS The species-dependence of this equilibrium is demonstrated in ultrafiltration studies that directly probe the bound Zt=*z free equilibrium, revealing Cu-PTSM to be 95% albumin-bound in human serum albumin (HSA) solution, but only 60% albumin-associated in dog serum albumin solutions (Mathias et al., 1995). The strength of this reversible Cu-PTSM interaction with HSA impairs the ability of 62Cu~PTSM to quantify myocardial perfusion by limiting the tracer's ability to passively diffuse into tissue at high rates of flow. Two related "second-generation" tracers have been identified, 62Cu-n-PrTS and 62Cu-ETS (Figure 2), that do not exhibit the inter-species variations in albumin binding that proved problematic with Cu-PTSM (John & Green, 1990; Mathias et al., 1995). One of these latter agents may remain suitable for clinical quantification of myocardial perfusion with PET and 62Cu. Clinical use of a 62Cu-tracer requires a convenient method for routine, repetitive, high-yield radiopharmaceutical synthesis using the eluate of a 62Zn/62Cu generator. The basic procedure for preparation of the 62Cu-BTS complexes, using the ionic 62Cu-generator, involves buffering the acidic 62Cu/HCI generator eluate, followed by mixing with the BTS ligand (Green et al., 1990). While previous work with 62Cu-PTSM relied on a fairly simple remote system for radiopharmaceutical synthesis (Mathias el al., 1991), 62Curadiopharmaceutical preparation has been further simplified by integration of reagent mixing operations into the 20x30x40 cm housing of a modular generator unit (Figure 4), available from Proportional Technologies, Inc. (Houston, Texas) (Haynes et al., 2000; Wallhaus et al., 1998). This modular generator can directly deliver the 62Cu-PTSM, 62Cu-n-PrTS, or 62Cu-ETS radiopharmaceuticals in sterile, pyrogen-free solution suitable for intravenous injection (Haynes et al., 2000; Lacy et al., 1995; Lacy et al., 1996; Wallhaus et al., 1998). The 62Cu2+ ion is selectively eluted from the shielded 62Zn/62Cu generator column in 0.2N HC1:1.8N NaCl using a multichannel peristaltic pump to regulate the rate of elution. A second channel on the peristaltic pump is used to mix the acidic eluate with two equivalents of a sterile aqueous sodium acetate (NaOAc) solution at the column outlet. A third peristaltic pump channel then delivers an ethanol solution of the BTS ligand into the acetate-buffered eluate stream, resulting in essentially quantitative formation of the 62 Cu-BTS radiopharmaceutical before the eluate reaches the outlet of the generator housing (Haynes et al., 2000; Lacy et al., 1995; Lacy et al., 1996; Wallhaus et al., 1998). This in-line synthesis methodology performs exceptionally well, delivering the 62Cu-BTS radiopharmaceuticals with radiochemical purity exceeding 98% at the generator outlet using only 1,5 ug of the BTS ligand per elution (Haynes et al., 2000; Lacy et al., 1995; Lacy et al., 1996; Wallhaus et al., 1998). The radiopharmaceutical synthesis time is simply the 40-second period required for generator elution. Such a modular 62Zn/62Cu generator system, nationally or regionally distributed from commercial medium-energy cyclotron facilities, may effectively support PET imaging centers as a source of short-lived radiopharmaceuticals for evaluation of tissue perfusion. This may become increasingly attractive, as regional pharmacy-based low-energy cyclotron facilities are becoming widely available to provide [18F]fluorodeoxyglucose (FDG) for complementary study of cerebral, myocardial, and/or tumor metabolism.

410

HANDBOOK OF RADIOPHARMACEUTICALS

Figure 4. Compact, modular 62Zn/62Cu parent/daughter generator designed to directly deliver 62Cubis(thiosemicarbazone) radiopharmaceuticals (Haynes et al., 2000; Lacy et al., 1995; Lacy et al., 1996; Wallhaus et al., 1998). (Photo from Proportional Technologies, Inc., Houston, Texas.)

VI. COPPER-LABELED HYPOXIA IMAGING AGENTS Hypoxic tissue is considered to be an important target for the detection of ischemia of the brain, heart and other tissues, as well as hypoxic tumors. Nitroimidazole compounds, originally developed as radiosensitizing agents, are of great interest because of their selective accumulation in hypoxic tumors (Evans et al., 2000; Kachur et al., 1999; Melo et al., 2000; Rasey et al., 2000; Stypinski et al., 1999; Su et al., 1999; Yamamoto et al., 1999; Yang et al., 1999). Nitroimidazole can be reduced and retained only in hypoxic tissue, but not in normoxic tissue. However, the problem with this class of compounds has been their low lipophilicity, resulting in low tissue accumulation. It has been demonstrated that Cu-PTSM is easily reduced by the electron transport enzyme of mitochondria (Taniuchi et al., 1995). The redox potential of Cu-PTSM is higher than that of NADH and the reduction of Cu-PTSM by mitochondria occurs almost quantitatively in an NADH-dependent manner. Interestingly, some Cu-BTS complexes having a more negative redox potential showed as high of a membrane permeability as Cu-PTSM, but were not retained in the brain or the heart. If these Cu-BTS complexes were selectively trapped by abnormally reduced mitochondria, like nitroimidazole, they may become a better candidate for hypoxia imaging.

CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS Cu-BTS

411

Cu-DDS H3Q, .CH3

r.r \ JL

^N-^ 1

S

S

N t

^3

(sal)2(2,2-Me2tn) ligand

Ri R2

PTSM PTSM2 PTSE ATSM ATSM2 ATSE ETSM ETSE

Me Me Me Me Me Me Et Et

H H H Me Me Me H H

R3

Me Me Et Me Me Et Me Et

R4 H Me H H Me H H H

(acac)2en

Figure 5, Cu-BTS and DDS complexes that have been evaluated as hypoxia imaging agents (Fujibayashi et al., 1999b).

Based on this hypothesis, various Cu-DTS complexes and other related compounds have been surveyed (Figure 5) (Dearling et al., 1998; Fujibayashi et al., 1999b). Interestingly, most of the Cu-complexes studied showed hypoxia selective reduction, although the enhancement levels varied (Fujibayashi et al., 1999b). These studies suggested that the nitroimidazole moiety is not essential for the uptake of a compound in hypoxic tissue. Among them, radiolabeled Cu-diacetyl- bis(N4-methylthiosemicarbazone) (Cu-ATSM) showed the most favorable characteristics as a hypoxia imaging agent (Dearling et al., 1998; Fujibayashi et al., 1997), The proposed retention mechanism of Cu-ATSM is shown in Figure 6. ~l Cu-(ll)-PTSM Membrane

T

Cu-(ll)-A TSM Easy penetration

"I Cu-dD-PTSM

e -^

!

Cu-(i)

i

Retention

I^

k

Blood Cell

I Cu-(ll)-ATSM

Normal XX Mitochondria ^T

e-

X

e "

^

I Cu-(l) |

Retention

Figure 6, Retention mechanism of Cu-ATSM in hypoxic tissue (Fujibayashi et al., 1997).

abnormally reduced Mitochondria by hypoxia

412 In the first clinical trial using generator-produced

HANDBOOK OF RADIOPHARMACEUTICALS 62

Cu in lung cancer patients (Takahashi et al., 2000), Cu-

ATSM clearly visualized cancerous lesions (Figure 7). Cultured cell studies indicated that the retention was closely correlated with levels of hypoxia in the tumors (Lewis et al., 1999b). Interestingly, basic research on the retention mechanism in tumor cells indicated that reduction site in tumor cells was different from that of hypoxic non-tumor tissues. In tumors, microsomal bioreductive enzymes have been highly expressed and they play a major role in the reduction of Cu-ATSM (Obata et al., 1999). Although the physiological role of microsomal bioreductive enzymes in tumor cells is not clarified yet, this finding might bring us new insights for the molecular-level change in electron transport in carcinogenesis, a seed for "imaging-based medical science".

Figure 7. PET and CT images of Cu-ATSM in a lung cancer patient. COPPER-LABELED PROTEINS AND PEPTIDES

COPPER-LABELED MONOCLONAL ANTIBODIES Monoclonal antibodies (mAbs) have been produced which bind to antigens present on a large number of tumor types. These mAbs have been labeled with radiometals for diagnosis and therapy of cancer, and have been reviewed many times during the last 10 years (Delaloye and Delaloye, 1995; McKeam, 1993; Schubiger et al., 1996; Waldmann, 1991; Zuckier and Denardo, 1997). Currently, indium-111 labeled DTPA-B72.3 (OncoScint™) and DTPA-7E11 .C5.3 (ProstaScint™) are approved for clinical use in the US. 64

Cu and 67Cu have been labeled to mAbs (both intact antibodies and antibody fragments) for diagnostic

imaging and radioimmunotherapy (RIT). DeNardo et al. (DeNardo et al., 1991) used a bifunctional chelator of TETA (bromoacetamidobenzyl-1.4,8,1 l-tetraazacyclotetradecane-N.N'.N".N'''-tetraacetic acid (BAT))

CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS

413

developed by Meares et al. (Moi et al., 1985) and 2-iminothiolane (2IT) (McCall et al., 1990) as the linker to bind the therapeutic radionuclide, 67Cu, to the mAb Lym-1 (Figure 8), and they evaluated this radiopharmaceutical in humans with non-Hodgkin's lymphoma. This pilot study (DeNardo et al., 1991), which was later confirmed in additional patients (DeNardo et al., 1999), demonstrated that the pharmacokinetics of the Cu-labeled Lym-1 were comparable to 131,I-Lym-1; however, 67,Cu has superior decay characteristics for RIT compared to 13hI. In patients with non-Hodgkin's lymphoma, the maximum tolerated dose (MTD) for 67Cu-2IT-BAT-Lym-l was determined to be 60 mCi/m2 for each of two doses (DeNardo et al., 1998). pH~8 30 min at 37° C

2-iminothiolane (2IT) NH,

mAb

6

2IT-mAb

*Cu-Acetate or citrate pH5.5 Incubate at RT for 30 min Purify by Centrifuged Gel Filtration Column

BAT-2IT-mAb

C. J

BAT

*Cu-BAT-2IT-mAb

Figure 8. Conjugation of BAT and 2IT with mAbs and subsequent labeling with copper radionuclides.

Pharmacokinetics of simultaneously injected 67Cu- vs 125I-labeled mAb 35, an anti-colorectal carcinoma mAb, was determined in six patients with colorectal cancer (Delaloye et al., 1997). 67Cu was labeled to mAb 35 via the macrocyclic bifunctional chelator 14N4 (or CPTA) (Smith-Jones et al., 1991). They found that the tumor uptake of 67 Cu-labeled mAb 35 was greater than the 125!I-labeled mAb, and the tumor-to-blood ratios were also higher for 0/67,Cu-mAb 35. Another intact mAb, C595, has been labeled with 67Cu and investigated as a potential RIT agent for local treatment of bladder cancer (Hughes et al., 1997). °4Cu-labeled BAT-2IT-1A3, an anti-colorectal monoclonal antibody was prepared as described in Figure 8. Cu-BAT-2IT-lA3 has been shown to be effective in PET imaging (Philpott et al., 1995) and also has potential for RIT (Connett et al., 1996). Patients with primary and metastatic colorectal tumors were imaged using PET and 64Cu-BAT-2IT-l A3, and it was found that 100% of primary tumors were detected, while only 40% of liver and lung metastases were visualized (Philpott et al., 1995). The lack of detection of metastases was likely due to higher background in liver and lung in these patients. Connett et al. compared 64Cu-BAT2IT-1A3 (2 mCi) with 67Cu-BAT-2IT-lA3 (0.4 mCi) in a colorectal tumor-bearing hamster model and 64

414

HANDBOOK OF RADIOPHARMACEUTICALS

demonstrated that both radiopharmaceuticals were able to cure 200-400 mg tumors in the animal model with no observable toxicity (Connett etal., 1996). In a later study, the MTD of ^Cu-B AT-2IT-1 A3 in the hamster model was determined to be 150 mCi/kg (Connett et al., 1999). Copper-64 and 67Cu have also been labeled to mAb F(ab')2 fragments, since F(ab')2 fragments are of lower MW (~110 kDa) compared to intact mAbs (~160 kDa) and clear the blood faster. Smith and colleagues investigated 67Cu-CPTA-SEN7 and its F(ab')2 fragments, and determined that improvements in blood clearance with the F(ab')2 fragments were overshadowed by the high accumulation in the kidneys (Smith et al., 1994). Subsequently, it was demonstrated with 67Cu-labeled CPTA-lA3-F(ab')2 that metabolism in the kidney occurred, and the radioactivity in the kidneys was found to be in the form of 67Cu-CPTA-lysine (Rogers et al., 1996). A peptide linker (triglycyl-L-p-isothiocyanato-phenylalanine) was used to attach CPTA to mAb chCE7 F(ab')2, and 67Cu-CPTA-Rl-chCE7 F(ab')2 had reduced uptake in the kidneys (Zimmerman et al., 1999).

COPPER-LABELED PEPTIDES The targeting of somatostatin receptors (SSR) in tumors has been a goal in cancer treatment and diagnosis for nearly two decades. Somatostatin is a 14-amino acid peptide which plays an inhibitory role in the normal regulation of several organ systems: 1) the central nervous system, the hypothalamus and the pituitary gland; 2) the gastrointestinal tract; and 3) the exocrine and endocrine pancreas (Reichlin, 1983a; Reichlin, 1983b). A large number of human tumors are also SSR-positive (Reubi et al., 1990). Somatostatin is unsuitable for in vivo use due to its very short biological half-life. Analogs of somatostatin have been developed, including octreotide (OC), that are highly resistant to enzymatic degradation (Bauer et al., 1982). Indium-111-labeled DTPA-OC is approved for human use in the U.S. and Europe as Octreoscan™, a diagnostic imaging agent for neuroendocrine tumors (Krenning et al., 1992). Octreotide has been conjugated to the macrocyclic chelator TETA for labeling with 64Cu (Anderson et al., 1995). 64Cu-TETA-OC (Figure 9) had high affinity for the SSR both in vitro and in vivo, and cleared primarily through the kidneys, with very low liver accumulation. 64Cu-TETA-OC was evaluated as a PET imaging agent for neuroendocrine tumors (Anderson et al., 2001). Preliminary results showed that 64CuTETA-OC was able to detect even more SSR positive lesions than the currently used, clinically approved agent, '"in-DTPA-OC (Pentetreotide) and gamma scintigraphy. 64Cu-TETA-OC significantly inhibited the growth of SSR positive tumors in rats (Anderson et al., 1998), and therefore has potential for both diagnostic imaging and targeted radiotherapy.

CHEMISTRY OF COPPER RADIONUCLIDES AND RADIOPHARMACEUTICAL PRODUCTS HOOC

-CONH-(D)Phe-Cys-Phe-(D)Trp

~\

H

°OC

\| ,N

|/ N

CONH-(D)Phe-Cys-Tyr-(D)Trp (OL)-Thr-Cys-Tfir-Lys

(OL)Thr-Cys-Thr-Lys

HOOC

"N '\

N I^

N

COOH

v| N

N.

-COOH

HOOC

TETA-OC

HOOC

TETA-Y3-OC

|/ CONH-(D)Phe-Cys-Tyr-(D)Trp N,

-CONH-(D)Phe-Cys-Phe-(D)Trp

HOOC

!

OH-Thr-Cys-Thr-Lys

OH-Thr-Cys-Thr-Lys COOH

HOOC

TETA-Y3-TATE

415

-COOH

HOOC

TETA-TATE

Figure 9. Structures of TETA-OC, TETA-Y3-OC, TETA-TATE and TETA-Y3-TATE for radiolabeling copper radionuclides for imaging and targeted radiotherapy of somatostatin receptor positive tumors (Lewis el al., I999a)

Somatostatin analogs have been prepared based on the structure of OC where tyrosine (Y) is substituted for phenylalanine (F) in the 3-position, and/or the C-terminal alcohol is replaced with a carboxylate (de Jong et al., 1998a; de Jong et al., 1998b). These substitutions have been made for 64Cu-TETA conjugates and have resulted in significantly higher SSR positive tissue uptake compared to 111 In-DTPA-OC and 64Cu-TETA-OC (Lewis et al., 1999a). The higher uptake of the Y3-TATE analogs over the Y3-OC and OC analogs is not entirely explained by differences in SSR binding affinity. In fact, DTPA-Y3-TATE has very similar binding affinity to DTPA-Y3-OC and DTPA-OC (de Jong et al., 1998b). Data published by de Jong et al. (de Jong et al., 19985), along with studies by Lewis et al. with 64Cu-labeled TETA-OC, TETA-Y3-OC, TETA-TATE and TETA-Y3-TATE (Figure 9) (Lewis et al., 1999a), showed that the kinetics of uptake of the TATE analogs by SSR positive cells is much faster than the OC analogs. The more rapid kinetics of uptake and internalization of the TATE analogs appeared to be the reason for the increase in uptake in SSR positive tissues. SUMMARY The past decade has seen a substantial increase in the availability of copper radionuclides for research in radiopharmaceutical chemistry. A number of radiotracers from these investigations have entered human clinical trials, for both diagnostic imaging and targeted radiotherapy. It is expected that such research efforts will continue into the foreseeable future, exploiting the rich coordination chemistry of copper, as well as the diverse nuclear properties of available radioactive isotopes of copper. ACKNOWLEDGEMENTS The authors would like to thank the National Cancer Institute [CA64475 (CJA) and CA70845 (MAG)] for financial support.

416

HANDBOOK OF RADIOPHARMACEUTICALS

GLOSSARY ATSM BAT BFC BTS CPTA DOTA DTPA EC EDTA ETS FDG h HSA keV MeV MTD n-PrTS NADH OC PCBA PET PTSM RIS KIT SCN-TETA SOD SPECT SSR TATE TETA Y3-OC Y3-TATE

diacetyl-^/sCA^-methylthiosemicarbazone) 6-[p-(bromoacetamido)benzyl]-l ,4-8-11 -tetraazacyclotetradecane-1,4,8,11 -tetrac acid Bifunctional chelator Bis(thiosemicarbazone) 4-[( 1,4,8,1 l-tetraazacyclotetradec-l-yl)methyl]benzoic acid 1,4,7,10-tetraazacyclododecane-l ,4-7,10-tetraacetic acid diethylenetetraaminepentaacetic acid electron capture (radioactive decay) ethylenediaminetetraacetic acid ethylglyoxal bis(thiosemicarbazone) [' 8F]-fluorodeoxyglucose Hours human serum albumin kilo-electron volts (103) mega-electron volts (106) maximum tolerated dose Az-propylglyoxal bis(thiosemicarbazone) nicotinamide adenine dinucleotide hydride octreotide 1 -[(1,4,7,10,13-pentaazacyclopentaadec-l -yl)methyl]benzoic acid positron emission tomography pymvaldehyde-biXA^-methylthiosemicarbazone) Radioimmunoscintigraphy Radioimmunotherapy 6-[/?-(isothiocyanato)benzyl]-l ,4,8,11 -tetraazacyclotetradecane Superoxide Dismutase single photon emission computed tomography somatostatin receptor Octreotate 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid tyrosine-3-octreotide tyrosine-3-octreotate

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13. CHEMISTRY APPLIED TO IODINE RADIONUCLIDES RONALD FINN Cyclotron Core Facility, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.

INTRODUCTION Iodine, which was discovered in 1811 by Bernard Courtois, occurs sparingly in nature in the form of iodides in sea water from which it is assimilated by various sea weeds, in brines from old sea deposits, and in brackish waters from oil and salt wells. The element is a grayish-black solid with a slightly metallic luster. It sublimes at atmospheric pressure and temperature resulting in a blue-violet gas with an irritating odor. To appreciate the recovery of radioiodine species from the various production target matrices and the subsequent strategies for radiolabeling compounds with various isotopes of iodine, an introduction to the classical chemistry of iodine is appropriate. Although iodine forms compounds with many elements, the most common compounds are the iodides of sodium and potassium, which find direct application in the radiolabeling of compounds. Iodine dissolves readily in nonpolar solvents such as chloroform, carbon tetrachloride or carbon disulfide resulting in beautiful violet solutions, but the element is only slightly soluble in water. Iodine compounds are important in organic chemistry, finding specific applications to medicine in both magnetic resonance imaging contrast enhancement reagents and radiopharmaceutical drugs. Iodine is the least abundant element of the principal elements in "standard man" comprising only 2.13 X 1C)–6 atom percent. The bulk of the approximately 30 mg of stable nuclide of iodine (1-127) contained within an adult individual is normally present in the thyroid gland (Myers et al., 1973). Lack of iodine is the cause of goiter in humans. A review of the chart of nuclides reveals that more than thirty radioisotopes of this element are known. The half-lives of the radioisotopes of iodine range from tenths of a second (1-142) to greater than 10 million years (I-129). Of these radioisotopes, primarily five nuclides are finding significant clinical applications. Specific characteristics (Browne et al., 1978; Kocher, 1981) for these radionuclides are outlined in Table 1. Several iodine oxides exist in nature, examples include I2O4,14O9,12O7 and I2O5. These compounds, when heated to temperatures above 100°C, decompose to yield I2O5 and iodine, or subsequently, elemental iodine and oxygen. There are several known oxo-acids of iodine solutions in various states of purity, which can be obtained by the reaction of the elemental iodine with water or aqueous bases (Schmeisser, 1963). However, the hypohalous acid of iodine theoretically formed from the reaction of iodine with water does not constitute a suitable method for its preparation due to unfavorable equilibria. In fact, of the hypohalous acids, HOI is Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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the least stable of the halogens and is unable to be isolated in the pure state (Cotton & Wilkinson, 1966). Hypoiodite ions can be produced, in principle, by dissolving iodine in base according to the general reaction: I2 + 2OH -» r + IO + H2O

A favorable equilibrium constant is reported at 30 for iodine, and the reaction is rapid. However, the hypoiodite ions tend to further disproportionate in aqueous basic solutions to produce the iodate ions: 3IO -> 21 + IO3

The equilibrium constant for 10- is reported at IO20. Since the rate of disproportionation of 10- is very fast at all temperatures, the overall reaction of iodine with base gives iodate (I03-) quantitatively (Cotton & Wilkinson, 1966) according to the equation: 3I2 + 6OH -> 51 + IO3 + 3H2O There is good evidence for the existence of cationic species of iodine, such as I+, I3+ and I5+ as well as I2Cl+, I 2 Br + and IC12+ ions under special conditions. (Cotton & Wilkinson, 1966; Awtrey & Connick, 1951; Arotsky ei al, 1961). Such ions are powerful electrophiles existing only in media with low nucleophilic properties. The electropositive iodo species is also classified as a powerful nucleophile and envisioned to be strongly solvated. The inter-halogen compounds, such as IC1, IC13 are quite reactive and behave as oxidizing agents. In their reaction with other elements, the inter-halogen compounds produce mixtures of the halides which are more or less readily hydrolyzed according to the equation: IX' f H2O -»• H+ + X' + HOI

NUCLEAR MEDICINE APPLICATIONS Radiotracer studies of the thyroid gland were the first clinically important procedures in nuclear medicine and provided a fundamental stimulus for the creation and early development of the entire discipline (Brucer, 1973; Atkins, 1969). Radioiodine became available from cyclotron production in the late 1930's on a limited basis. With the development of the atomic reactor in the 1940's, availability of radioiodine improved and the United States Atomic Energy Commission began providing 1-131 for widespread clinical use shortly after the end of World War II (Beierwaltes, 1979). Moreover, the Journal of the American Medical Association published a report in 1946 on the use of iodine131 to "remove" several cancerous tumors that had spread from the original tumor site in a patient's thyroid gland. The thyroid tumor already had been surgically removed, but clearly that had not stopped the spread of disease. Dr. Seidlin and his colleagues from Montefiore Medical Center in New York reported the treatment. By 1964, the Atomic Energy Commission had licensed more than one thousand United States physicians to utilize radionuclides in their practice of medicine and authorized more than twelve hundred medical institutions to handle radionuclides. In 1971 the American Medical Association recognized nuclear medicine as a specialty discipline. By 1996, it was estimated that one half of the hospitals within the United States had

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nuclear medicine services, and more than eleven million individuals had received a radiopharmaceutical for either diagnostic or therapeutic purposes (Wagner Jr, 1996). SPECIFIC RADIOISOTOPES OF IODINE Iodine-127 is the only stable isotope of iodine, while there are in excess of 30 radioactive isotopes of iodine reported. Approximately ten of these isotopes have been evaluated for biomedical applications. Those isotopes of iodine with mass number greater than 127 undergo beta-minus decay with the exception of 1-128, which also decays by electron capture 6.4% of the time. The beta particles are emitted with a continuous spectrum of kinetic energy up to a maximum value characteristic of the particular radionuclide. The maximum beta energy is an important consideration as it defines the distance or range in soft tissue over which the energy and radiation dose will be deposited. Radioisotopes of iodine with mass number less than 127 undergo decay by electron capture and by positron emission. The absence of paniculate emissions in the neutron-deficient nuclides significantly reduces the radiation absorbed dose to the individual. Iodine-131 is the ''classic" radioisotope of iodine from the standpoint of its medical applications. Its sodium salt was used almost exclusively for two decades for percent thyroid uptake measurements and imaging applications. Its current use has been somewhat restricted due to the relatively high radiation dose to the thyroid gland and its less than ideal photon characteristics for patient imaging with gamma-scintillation cameras or available collimation due to either relatively low crystal efficiency and high septal penetration. Iodine-123 may be represented as the "ideal" radioisotope of iodine particularly for thyroid-imaging applications and for uptake studies (Thrall, 1990; Stocklin, 1977; Nozaki, 1983). The half-life of 1-123 is 13.3 hours, which is satisfactory for clinical imaging although somewhat restrictive when considering the logistical problems in shipment of the radionuclide. Iodine-124 represents a change in philosophy by the nuclear medicine community. Originally, this radionuclide was seen as a detriment in that it was a radionuclidic impurity in the commercial preparations of iodine-123. Its presence in iodine-123 resulted in increased radiation dose to the patient and was problematic due to the high-energy photons of 602 KeV and 722 KeV. These photons lead to degraded images caused by collimator septal penetration and scattering. Current interest in appropriate production methods and its clinical applications are the result of the increased emphasis being concentrated on the role of positron emission tomography in the future evolution of the nuclear medicine specialty. Iodine-122 is a generator-produced radionuclide with a half-life of 3.6 minutes. It decays by positron emission (77%) and has a characteristic photon of 0.564 MeV (18.4%). Its application has been proposed for the measurement of rates of metabolism and short-term biodistribution imaging within selected organ blood pools. Enzymatic catalyzed reactions have been shown to provide specific radiolabeled Pharmaceuticals (Mausner et al., 1984). The production and availability of the parent xenon-122 is critically dependent upon high energy accelerator's irradiation schedules and as such, the availability of the parent will influence further development and applications of this of iodine.

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As can be appreciated from this brief introduction, there is a long history of radionuclide use in medicine and the number of applications in this area is increasing constantly with the development and implementation of new imaging techniques and radiopharmaceutical design. Radiopharmaceuticals account for the principal application of radionuclides within Nuclear Medicine Services, while radioimmunoassay for in vitro tests utilize primarily iodine-125 with lesser application of tritium and iron-59. Several radiopharmaceuticals in routine application contain radioisotopes of iodine (Maltly, 1994; Mather, 1994; Owunwanne et al., 1995). Applications of radiation fields include metabolic radiotherapy, remotely controlled cobalt therapies and brachytherapy. Metabolic radiotherapy primarily is concerned with treating hyperthyroidism and thyroid cancers. This treatment is often a cooperative service between nuclear medicine departments and radiation therapy departments. Brachytherapy utilizes a variety of radionuclides, such as indium-192, palladium-103, and iodine-125, encapsulated in seed form for generation of a localized radiation therapy of the patient in which the seeds are implanted. It is an expanding application for use of radionuclides applied to organ specific therapeutic radiation treatments. In addition to the applications of radioiodine to thyroid studies, an early appreciation of the renal excretion of specific radiographic contrast agents prompted investigations into iodinated radiopharmaceutical preparations for the assessment of renal function. The first reported clinical use of radioactive tracers to measure quantitative differences in renal function occurred in 1956 when G.V. Taplin and co-workers used I131 labeled Diodrast and collimated external scintillation detectors to evaluate the time-related clearance functions of the kidney. However, 1-131 Diodrast, like many of the other radioiodinated contrast media-type renal agents, proved unsatisfactory since a significant fraction of the injected dose also underwent non-renal excretion (Ponto et al., 1990). Experimental observations of Pressman (Pressman & Korngold, 1953) published in 1953 demonstrated the application of radiolabeled antibodies to localize and visualize tumors. This area of investigation continues today to receive increased attention from radiochemists for potential clinical applications of iodine radioisotopes to radioimmunoimaging and radioimmunotherapy by incorporation of the iodine nuclide into the chemical structure of various peptides and monoclonal antibodies. For several decades, radiolabeled antibodies have undergone evaluation for their potential to detect a variety of tumors and occult masses (Waldman, 1991; Kemshead & Hopkins, 1993). Earliest experiments had suggested that antibodies might deliver radionuclides specifically to target tissues like "magic bullets." However, preclinical and clinical experiences with radiolabeled antibodies have not realized the expectations regarding specificity and sensitivity of tumor localization with these agents. In fact, quantitative experiments have demonstrated that only a small proportion of the total administered activity associated with radiolabeled antibodies localizes at tumor sites (Mach et al., 1980; Colcher et al, 1987). It appears that several factors may limit the current usefulness of intact radiolabeled antibodies for tumor detection. Such factors as tumor biology and immunology, antibody pharmacology and pharmacokinetics, and the resultant radio immunochemical properties of anti-tumor antibodies appear to be important properties in the radioimmunodetection of tumors. The resultant immunoreactivity of a radiolabeled antibody may be possibly its most important biological feature for delivery of a therapeutic dose to the tumor site (Chilton et al., 1990). All labeling procedures pose a risk in decreasing the biological activity of the target protein through chemical alteration. It has been

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documented that the immunoreactivity of therapeutic antibody radioiodinated complexes is decreased exponentially as the iodine-to-fragment ratio is increased (Nikula, 1998; Ferens et al., 1984 ; Larson et al., 1984; Matzku et al., 1987). Advancements in immunochemistry resulted from a better understanding of tumor biology and immunochemistry. The discovery of monoclonal antibody technology (Kohler & Milstein, 1975) has led to a wide array of immunoglobulin reagents that have shown specificity for various tumor types. Based upon both animal and human data, tumor-associated antigens in appropriate cellular expression have proven to be the targets for radiolabeled antibodies. Despite such attraction between specific tumors with specific antibody reagents, investigators have found that most tumors are quite heterogeneous in their expression of tumor-associated antigens and as such concern exists that cells expressing low levels of antigen may escape detection with application of radiolabeled antibody (Burchiel et al., 1982). APPROACHES TO RADIOLABELING WITH ISOTOPES OF IODINE It is beyond the scope of this chapter to give a complete review on all iodination procedures suitable for the radiolabeling of compounds, proteins or antibodies but rather to point out the subtleties and similarities of various chemical procedures that have become well established and are applicable to a variety of applications. Most often the chemical form of the starting radioiodine is that of the iodide anion at the "nocarrier-added" concentration. It is important to point out some difficulties which occur when dealing with species in carrier-free form. Losses in the activity which can occur particularly in chromatographic procedures, are common and changes in the chemical oxidation state of the iodine species are observed which are dependent upon the environment. The exact oxidation state of a "carrier-free" iodine species under the condition of the analytical quality control is questionable, and the stabilization or the protection of the halide is often chemically necessary through the addition of reducing or stabilizing agents such as thiosulfate, EDTA or the addition of "carrier" iodide when the procedure can tolerate the conditions (Stocklin, 1977). Synthetic approaches for the radioiodination of compounds generally follow established, conventional chemical methods. Direct methods involve the use of Chloramine-T (Hunter & Greenwood, 1962), lodogen (Fracker & Speck 1978) and lactoperoxidase (Marchalonis, 1969). Indirect methods which have been utilized include conjugation of the radioiodinating reagent, such as Bolton-Hunter reagent (Bolton & Hunter, 1973), N-succinimidyl para-iodobenzoate (Zalutsky & Narula, 1987) and N-succinimidyl 3-hydroxy-4-iodobenzoate (Vaidyanathan et al., 1997), methyl p-hydroxybenzimidate hydrochloride (Wood et al., 1975), fluoresceine isothiocyanate (Gabel & Shapiro, 1978), and N-chloro-iodotyramine (Holowka, 1981); regio-specific iodination reactions, addition of iodine monochloride to double bonds, replacement of the diazo groups, and enzymatic methods using lactoperoxidase (Thorell & Johansson, 1971; Miyachi et al., 1972; Krohn & Welch, 1974) to mention but a few. The biologically active compounds which require extremes in high specific activity are often synthesized by displacement reactions. Syntheses by exchange reactions must be undertaken with caution as it is frequently necessary to employ "carrier-added" iodide to achieve a favorable radiochemicai yield. Furthermore, impurities in the organic compound may be iodinated, and it is essential to ensure the purity of all reagents, and the attention to quality control analyses of the final products that are

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achieved. The most objectionable impurity in radioiodine-labeled compounds is often the radioiodide. The radioiodide may be formed in aqueous solution by radiation decomposition or by chemical decomposition during storage. The conjugation labeling method was developed to overcome problems of iodination damage associated with exposure of proteins to oxidizing and reducing reagents. The conjugation method has also found application in labeling enzyme preparations. In many instances, it can be removed as it is being formed by addition to the reaction vessel of a suitable ion-exchange resin or radiation protection reagent. The literature was reviewed through 1982 by Seevers and Counsell (Seevers & Counsell, 1982) addressing radioiodination reactions of small molecules by various iodinating reagents and in 1992 by Dewanjee (Dewanjee, 1992). A description of the preparation, biologic behavior and quality control procedures of some of the more commonly applied radioiodinated techniques, including catalytic, enzymatic and electrolytic has been published (Bolton, 1985) and the applications to which the radiopharmaceuticals radiolabeled with iodine radioisotopes appear in numerous literature articles which generally include the radiopharmaceutical chemistry procedures employed. EXCITATION LABELING PROCEDURES Hot atom chemists have availed themselves of excitation labeling by utilizing energetic, highly reactive radioactive species formed from induced nuclear reactions or nuclear decay processes to react with a substrate. The iodine-123 radiolabeling of specific compounds could be achieved from the interaction of the compound with the parent Xe-123. Beta decay gives rise to different types of excitation of the daughter atom including a change in the chemical identity, a change in kinetic energy from the electron and neutrino recoil and a change in charge state. It has been reported that iodine ions resulting from the decay of xenon-123 parent receive some kinetic energy resulting from neutrino emission (maximum recoil energy 34 eV) and from P* emission (maximum recoil energy 20 eV) (Stocklin, 1977). When the decay is allowed to occur in the presence of an organic substrate the iodine species can undergo rapid reactions in various oxidation states, i.e., electrophilic, homolytic or nucleophilic substitution processes. The resultant iodination induced from the decay of Xe-123 frozen on serum albumin was initially reported by Welch (Welch, 1970) at 80% radiochemical yield of organically bound iodine while Lambrecht and Wolf reported a 20% yield for labeling of indocyanine green via the decay of Xe-123 adsorbed at the dye surface at 77 K (Lambrecht & Wolf, 1973). In general, the direct decay-induced labeling of solid biomolecules has resulted in poor radiochemical yields due to the finite solubility of the parent Xe-123 in organic solids and subsequent labeling restricted to the surface of the solid substrate. Moreover, the decay induced labeling is inherently nonspecific with both hydrogen and halogen atoms being replaced (Stocklin, 1977). Improvements to the approach have resulted from preparation of iodinating reagents that can subsequently serve as the principal reagent for the labeling procedure. Such has been the case in the preparation of carrierfree iodine monochloride via the decay of Xe-123 in chlorine and the preparation of reactive iodination reagent produced with decay of Xe-123 on solid potassium iodate. The latter method produced good radiochemical yields with aromatic systems. However, there are some precautions expressed in the application of this method. (Lambrecht et al., 1972 ; Stocklin, 1977).

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HALOGEN EXCHANGE LABELING One of the more conventional approaches for the introduction of radioiodine into a molecule has been the halogen exchange reaction. This technique has been reported for both an isotopic and non-isotopic exchange with radioiodine at the "no-carrier-added" concentration level. The synthetic approach could be either nucleophilic via iodide or electrophilic via positive halogen species. Isotopic exchange has been reported in molten organic systems in which halogen exchange in solution could not conveniently be achieved (Elias et al., 1973). The technique for iodine radiolabeling is based on the isotopic iodine exchange occurring in the molten state of an organic substrate with "carrier-free" sodium iodide. The method has been used to prepare various radioiodinated products including p-iodophenylalanine, o,m,p-iodohippuric acid, and radiolabeled iodo estradiols. The limitation on this method rests in the necessity of melting the substrate or having a low melting analogue of the compound that can be melted without decomposition (Stocklin, 1977). Halogen exchange in aromatic systems is often difficult to accomplish, however, the nucleophilic exchange of iodine for bromine in aliphatic compounds via a nucleophilic reaction or melt technique has the potential to yield iodinated radiolabeled compounds. A recent example of the radioiodine for bromine exchange reaction applied to the preparation of 1-124 labeled 10-desmethoxy-10-iodocolchicine (Finn et al., 2001) is outlined in Figure 1. In special cases, exchange labeling on chromatographic supports and/or ion exchange resins can be performed (Boothe et al., 1985). The technique has not been routinely applied to radioiodination of radiopharmaceuticals commonly utilized in nuclear medicine procedures. IODINATION REACTIONS UTILIZING CHEMICAL OXIDIZING REAGENTS The classical sources of radioiodine for the labeling of organic molecules have included Nal, I2, HI, IC1, and organic iodide derivatives such as alkyltriphenoxy phosphonium iodide and nitrogen aromatic compounds. Sodium iodide appears to be the simplest and most versatile source of iodinating reagent. It can be used directly, in nucleophilic substitution for halogen or phenols in both aliphatic or aromatic compounds, regioand stereo-specific nucleophilic substitution reaction with metallic aromatic compounds and substitution for diazonium salts (Dewanjee, 1992). Elemental iodine formed by oxidation of sodium iodide in acidic medium with hydrogen peroxide, potassium iodate, sodium nitrite, or a variety of other oxidizing agents in neutral buffer can be used for radioiodination of organic compounds by the addition to double bonds or the substitution for hydrogen or halogen. The main drawback of this technique for nucleophilic radioiodination with iodine is that the oxidizing agent may potentially degrade the molecule to be labeled. Such has been the experience with the radiolabeling of various proteins and monoclonal antibodies. The techniques for radioiodination of proteins and peptides have improved significantly. The early reaction conditions reported by Pressman (Pressman & Keighley, 1948) utilized a mixture of iodide/iodate to achieve the radioiodination. McFarlane (McFarlane, 1958) popularized the IC1 technique in 1958. Hunter (Hunter &

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Greenwood 1962) introduced the procedure involving chloramine-T in 1962 and Rosa (Rosa et al., 1964) reported the electrolytic labeling method for sensitive proteins. Marchalonis (Marchalonis, 1969) reported the use of lactoperoxidase in 1969 and Bolton (Bolton & Hunter, 1973) introduced the method in which the radioiodine is covalently bound to lysine residues of proteins. This conjugation technique provides a complementary method for the radiolabeling for those macromolecules that do not possess either tyrosine or histidine residues or, alternatively, proteins that are sensitive to strong oxidants. Fraker (Fraker & Speck, 1978) introduced lodogen, which through modifications, has become the technique of choice for the radiolabeling of most proteins. In spite of the variety of methods reported, sodium iodide remains the source of radioiodine and the exact chemical nature of the reactive iodine species remains conjecture in some of the methods. Despite the numerous methods of radioiodination of proteins, they can be divided into two broad classifications, i.e., direct radiolabeling and conjugation labeled. Commonly radioiodination of proteins formulated by "in-house" methods for approved clinical procedures generally rely upon the reagents chloramine-T and/or lodogen due to availability of reagents and finite chemical reaction conditions. On occasion the electrolytic or enzymatic procedure, such as lactoperoxidase, are undertaken. In the lactoperoxidase reaction, carrier-free radioiodine and the compound to be radiolabeled are allowed to mix with nanomolar quantities of hydrogen peroxide. This technique has been effective in producing radiolabeled proteins in good yields with high specific activity and retention of immunoreactivity. Strong ring activating groups, such as amino or hydroxy groups, on the aromatic ring influence the substitution reaction. The resultant charge distribution on the phenyl ring affects the rate, extent and site of the ring iodination (Nikula, 1998). In order to appreciate the subtleties of radioiodination techniques of small molecules and various proteins, it is important to recognize several influences including the effect of solvent on radioiodination reactions; the strength of oxidizing reagents on the radioiodine; and the effects of other reactive species induced during the synthetic procedure. Various aspects of these reaction influences have been reviewed (Hughes, 1957; Ramachandran, 1956; Counsell & Ice, 1975; Seevers & Counsell, 1982; Koziorowski, 1998). It is also clear that the overall stability of radioiodinated antibodies is dependent upon the method of iodination used and the particular antibody that is being radiolabeled (Chilton et al., 1990). It is curious that despite several decades of radioiodination reactions involving biomedical studies, the chemistry of these reactions remains incompletely understood. It is generally accepted for the radiolabeling of monoclonal antibodies that the iodination reaction is predominantly occurring at the tyrosine and to a lesser degree, the histidine amino acid sites. The degree of iodination of these amino acids depends upon the structure of the protein, i.e., the amino acid residues on the surface of the protein will undergo iodination readily (Nikula et al., 1995) and the reagent utilized for oxidation may affect the overall radiochemical yield and the resultant product's biologic or immunologic activity. In the development of macromolecule radioiodinations, the methods were initially based upon the reactions of elemental iodine with phenolic compounds. To obtain the elemental iodine from commercially available sodium radioiodide various oxidizing reagents were added and extraction of the elemental iodine into nonpolar solvents. Some of the various reagents used to oxidize the iodide included nitrous oxide (Yalow & Berson, 1960), ammonium persulfate (Gilmore et al., 1954), hydrogen peroxide (McFarlane, 1956). ferric sulfate (Stadie et al., 1952), iodide-iodate system (Francis et al., 1951), and chromic acid (Heideman Jr. et

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at., 1965). In the application of this technique, generally sodium iodide was allowed to react with the oxidizing agent, and a stream of air or nitrogen gas was used to purge the volatile radioiodine species from solution into a reaction vessel containing the solvated protein. Iodine monochloride with radioactive sodium iodide was introduced for protein labeling in 1956 (McFarlane, 1956). Studies involving the iodination of phenol, substituted phenol and tyrosine with IC1 suggested that the mechanism of labeling was similar to that with elemental iodine, that is, electrophilic attack on the phenoxide ion followed by slow loss of a proton (Redshaw & Lynch, 1974). The suggested electrophilic species were H2OI+ or HOI at low pH and IC1 at the higher pH of the solvent (Helmkamp et al, 1967; Rao & Padmanabha, 1981). Chloramine-T immobilized on polystyrene beads and iodogen absorbed onto beads (Markwell, 1982) are commercially available. The advantage of the oxidant being absorbed onto beads rests in the fact that following the labeling process, the beads can be readily separated from solution eliminating the need for addition of a reducing reagent. Various catalysts have been investigated in an effort to improve the labeling efficiency of the radioiodide exchange reaction and to decrease the length of time for the reaction by employing various metallic catalysts. Such catalysts as copper, cuprous chloride, polymer supported phosphonates, silica gel, and various crown ethers have been evaluated (Dewanjee, 1992). The phosphonates appear to catalyze the exchange reaction between radioactive Nal and alkyl halides, while cuprous salts have been reported to catalyze the reaction between aromatic iodides and radioiodide in DMS, although the role of copper remains inconclusive. Attempts to improve the immunoreactivity fraction of the monoclonal antibody radiolabeled with iodine were successful when the antigen-binding site was occupied by the antigen during the labeling procedure (Van den Abbeele et al., 1991), REGIO-SPECIFIC REACTIONS WITH RADIOIODINE The use of radioiodinated physiologically active compounds having applications in tracer kinetic modeling, biochemistry, radiotherapy and medical imaging has been well documented (Heindel, 1978). A particularly desirable radioisotope of iodine for this application has been iodine-123 for the clinical application with single-photon emission tomographic imaging. This is due to the radionuclides emission of 159 keV photons (85%) and its reasonable half-life of 13.13 hours. Recently, with a restricted availability of iodine-124, interest in the radiolabeling of biomolecules has increased employing iodine-124 as the source of radioactivity. This radioisotope, having a convenient 4.2 day half-life, has a decay scheme which consists of electron capture and positron emission. Several techniques have been reported for the regio-specific iodination of organic molecules. The older classical approaches involved the introduction of the radioiodine onto ring compounds such as benzene, naphthaline, or other aromatic compounds which initially contained primary amino groups by utilization of the Sandmeyer reaction. When the functional groups were stable or were protected during the subsequent reaction process, the primary amino group was diazotized and subsequently displaced with radioiodine. The Gatterman reaction is a modified reaction using dediazotization which incorporates a copper bronze catalyst

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(Furniss et at., 1978) and the Wallach reaction which results in the formation of triazenes has found application for radio halogenation in aromatic compounds (Emeleus & Anderson, 1960). In an attempt to circumvent chemical and in vivo deiodination of specific radiopharmaceuticals, the preparation of novel organometallic precursors has been researched. Such precursors utilize a variety of metals including boron, mercury, tin, thallium, silicon and germanium to incorporate radioiodine onto vinylic, aromatic and heteroaromatic groups using iododemetallation reactions (Kabalka & Varma. 1989). The precursor metallic reagent can be stereospecifically introduced into the molecule at the latter stages of synthesis and in the presence of a great variety of functional groups. Utilizing these synthetic approaches, yields of the radioiodinated compounds are increased by reducing losses due to radioactive decay and subsequent chemical recovery/handling losses. The iododemetallation reactions have been successfully adapted to a "no-carrier-added" scale for the preparation of high specific activity products. Such is the example for the radioiodinated 2'-fluoro-2'-deoxy-l-p-D-arabinofuranosyl-5-iodo-uracil (FIAU), an effective probe for imaging the expression of the "marker/reporter gene", HSVl-tk (Tjuvajev et al., 1996). The synthesis of the iodostannylation precursor was undertaken in our laboratory in an effort that successfully resulted in improved radiochemical yields of radiolabeled FIAU utilizing 1-124 (Balatoni et al., 1998). The oxidizing reagent was hydrogen peroxide in glacial acetic acid. The reaction scheme leading to the radioiodinated pharmaceutical is outlined in Figure 2. RADIOIODINATION VIA ORGANOBORANES Organoboranes have been extensively used as precursors in radioiodination reactions. The syntheses of radioiodinated iodoalkyl, iodovinyl, and iodoaryl derivatives by reaction of either trialkyl borane or organoboronic acid intermediates with sodium radioiodide in the presence of a mild oxidant such as chloramine-T has been used to achieve radiochemical yields on a "no-carrier-added" scale. The use of organoboranes for radioiodination was first reported by Kabalka (Kabalka & Gooch, 1981; Kabalka et al., 1982). It has been suggested that organoboronic acids are the most valuable borane precursors for radioiodination because they are generally stable substances which can be handled without utilizing anhydrous or inert techniques. Synthetic methods are available for the preparation of vinyl and arylboronic acids of alkyne and aryl molecules that contain a variety of physiological active functional groups and for fatty acid analogues all of which are reactive with electropositive radioiodine to yield the desired radiotracer (Goodman et al., 1992). RADIOIODINATIONS VIA ORGANOSTANNANES Organostannanes are also versatile intermediates for the preparation of functionally substituted radioiodinated iodovinyl, iodoaryl and iodothienyl derivatives. It is reported that the tin carbon bond can be cleaved by molecular halogen (Bazantera/., 1965). The vinylstannanes undergo rapid radioiododestannylation on a "no-carrier-added" scale similar to that of the vinylboranes when treated with labeled sodium iodide and a mild oxidant. Iodovinyl carbohydrates and radioiodinated fatty acids have been reported from the iododestannylation reaction (Goodman et al., 1986; Goodman et al., 1991a; Goodman et al., 1991b; Goodman et al., 1989). It has also been documented (Goodman et al., 1992) that metallation of thiophene

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with alkyllithium regio specifically introduces the lithium metal into the 5-position of thiophene ring system which was transmetallated with trialkyltin halide and successfully iodinated. RADIOIODINATIONS VIA OTHER DEMETALLATION REACTIONS Organic thailates such as bis-(trifluoroacetoxy)thallium derivatives are excellent reagents for the specific introduction of radioiodine into aromatics systems. Some successes have been reported in the radiolabeling of phenyl and thienyl fatty acids utilizing this reagent. (Knapp et al., 1986). The thallation of monosubstituted phenylalkyl compounds with thallium(III) trifluoroacetate (TTFA) in trifluoroacetic acid occurs predominantly at the para-position whereas thallation of 2-substituted thiophene derivatives with TTFA in acetonitrile directs the thallium group into the 5-position. Generally, the aromatic thallium reagents are prepared in situ and are not isolated (Goodman et al., 1992). In an analogous reaction to that of the tin carbon bond iodination, Wilbur (Wilbur et al., 1982) has reported the preparation of iodinated aromatic compounds in high radiochemical yield by allowing a mixture of aromatic trimethyl silanes, radioiodide, and tert-butyl hypochlorite as oxidant in acetic acid. The radioiodination of various ligands for preparation of various imaging agents for specific receptors is an active area of manufacture. Of the various demetallation reactions presented, the application of the iododestannylation reaction to achieve the high specific activity, stereo- and regio-specificity of the desired compounds appears prominently in the current literature (Van Dort & Hagen, 2001; Hamill et al,, 2001a; Hamill et al., 2001 b). STRUCTURAL INTEGRITY OF RADIOIODINATED COMPOUNDS The determination of resultant structure of the labeled compound is surmised based on similar response to quality control processes that include the stable iodinated species obtained through a conventional chemical synthesis. The specific activity, that is, the concentration of iodide utilized for a radioiodination procedure is commonly insufficient to obtain mass spectroscopic information directly on the radioiodinated product. Moreover, the radiolabeled compound has an inherently finite shelf life (Chakrabarti et al., 1996). The radioiodinated product compound is identified by comparison to the similarity in responses to various analytic methods of the authenticated stable compound. Emphasis is being increasingly placed upon the structural configuration for interpretation of the in vivo distribution and metabolism of radiolabeled compounds. In this regard and with special consideration to radioiodination of antibodies and/or biomolecules, several criteria are necessary for development of a successful radiopharmaceutical product. These criteria include a labeling procedure that preserves the immunoreactivity of the antibody; the pharmaceutical product that results must be stable to rapid chemical breakdown; and concentrations of nuclidic impurities that may affect the activity of the radiolabeled antibody must be eliminated during the processing procedure.

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CONCLUSION Isotopes of iodine have for decades played a fundamental role in biomedical research and in clinical practice. The diversity of decay schemes available for the various isotopes of iodine have allowed a wide range of medical applications. Although radioiodination is a widely used technique for labeling a variety of materials, there appear to be four radioisotopes of iodine that have found significant applications in both research studies and clinical applications. Iodine-125 is a convenient radionuclide for laboratory studies and for radioimmunoassay, frequently iodine131 has been applied to radiopharmaceuticals for diagnostic and systemic radionuclidic therapy (Wilbur, 1992), iodine-123 bound to various drugs has found a wide application for diagnostic imaging (Qaim & Stocklin, 1983) and iodine-124 is finding applications for imaging and kinetic modeling evaluation using positron emission tomography, a rapidly developing component for the nuclear medicine service. Although advocated that the use of radioiodine was advantageous compared to trace metals for labeling of antibodies, the loss of radioiodine from monoclonal antibodies resulting from data on a number of dual labeling studies has now led to a strategy for development of various radioiodinating conjugate reagents in which the radioiodine is bound to a non-phenolic benzene ring (Zalutsky & Narula, 1987; Zalutsky, 1988; Koziorowski. 1998). In spite of specific limitations, radioiodinated tracers have been and will continue to be used in targeted therapies for the evaluation of the metabolic rates of plasma proteins and the binding of tracer compounds to cell membrane receptors. Such is the current situation with the application of various radiolabeled Pharmaceuticals including monoclonal antibodies that are undergoing evaluation for their sensitivity and specificity in the detection of primary and metastatic sites of malignant disease. Many modifications to synthetic techniques for iodination have been reported over the past fifty years with numerous references being provided for biomedical applications. Literature references related to production and to the separation of the radioisotopes of iodine from both reactors and cyclotrons targets; the variety of synthetic procedures for radiolabeling of both large and small molecules and the purification of the radiolabeled product from the reagents utilized in the procedure; the methodologies for measurement of biologic activity and the retention of immunoreactivity, sterility and apyrogenicity, all have impacted on the synthetic approaches to the radioiodination of various simple and complex chemical compounds that have been reported.

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REFERENCES Arotsky L Mishra HC and Symons MCR (1961) Unstable intermediates. Part XIII. Iodine cations in solution. J. Chem. Soc., 12–18. Atkins H (1969) Radionuclides and the endocrine system. In Handbook of Radioactive Nuclides, Wang Y (ed), Chemical Rubber Company, Cleveland, pp. 395-412. Awtrey AD and Connick RE (1951) The absorption spectra of I2, I3", I", IO3", S4O6" and S2O3. Heat of the Reaction I3 -> I2 + 1". J. Am. Chem. Soc., 73, 1842–1843. Balatoni, J, Finn R, Blasberg J, Tjuvajev J, and Larson S (1998) Production and quality assurance of cyclotron produced iodine-124 from enriched tellurium targets. In Applications of Accelerators in Research and Industry, Duggan JL and Morgan IL (eds), AIP Conference Proceedings 475, New York, pp. 984-986. Bazant V, Chvalovsky V, and Rathousky J. (1965) Organosilicon Compounds, Vol. 1, Academic Press, New York. Beierwaltes WH (1979) The history of the use of radioactive iodine. Sem. Nuc. Med., 9, 151–55. Bolton AE (1985) Radiation Technology, Amersham Corporation, Arlington Heights, pp.41–47. Bolton AE and Hunter WM (1973) The labeling of proteins to high specific radioactivities by conjugation to a

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I-containing acylating agent. Biochemical Journal, 133, 529-539. Boothe TE, Finn RD, Vora MM, Emran AM, Kothari PJ and Kabalka GW (1985) Radioiodinations of organic molecules on silica gel surfaces. J. Labelled Com. Rad., 22, 1109–1122. Browne E, Dairiki J and Doebler R (1978) Table of Isotopes 7th Edition, Lederer CM and Shirley V (eds), J Wiley and Sons, New York, pp. 592-659. Brucer M (1973) Thyroid Radioiodine Clinical Testing, 2nd Edition, Mallinckrodt Chemical Works, St. Louis, pp.5–15. Burchiel SW, Martin JC, Imai K, Ferrone S and Warner NL (1982) Heterogeneity of HLA-A,B,Ia-like and melanoma-associated antigen expression by human melanoma cell lines analyzed with monoclonal antibodies and flow cytometry. Cancer Research, 42, 4110–4115. Chakrabarti MC, Le N, Paik CH, De Graff WG and Carrasquillo JA (1996) Prevention of radiolysis of monoclonal antibody during labeling. J. Nuc. Med., 37, 1384–1388. Chilton HM, Burcheil SW and Watson Jr NE (1990) Radiopharmaceuticals for imaging tumors and inflammatory processes: gallium, antibodies and leukocytes. In Pharmaceuticals in Medical Imaging, Swanson DP, Chilton HM and Thrall JH (eds), MacMillan Publishing Company, New York, pp. 564598. Colcher D, Esteban JM, Carrasquillo JA, Sugarbaker P, Reynolds JC, Bryant G, Larson SM and Schlom J (1987) Quantitative analyses of selective radiolabeled monoclonal antibody localization in metastatic lesions of colorectal cancer patients. Cancer Research, 47, 1185–1189. Cotton F and Wilkinson G (1966) Advanced Inorganic Chemistry, Interscience Publishers, New York, pp. 559-590. Counsell RE and Ice RD (1975) The design of organ imaging radiopharmaceuticals. In Drug Design, Vol.6, Ariens EJ (ed), Academic Press, New York, p. 171. Dewanjee MK (1992) Radiation: Theory, Practice. Biomedical Application., Kluwer Academic, Boston, pp. 69-194,

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Emeleus HJ and Anderson JS (1960) Modern Aspects of Inorganic Chemistry, 3rd ed., rev., Routledge and Kegan Paul, London, p. 358. Elias H, Arnold C and Kloss G (1973) Preparation of I3ll-labelled m-iodohippuric acid and its behaviour in kidney function studies compared to o-iodohippuric acid. Int. J. Appl. Rad. Isot., 24, 463–469. Ferens JM, Krohn KA, Beaumier PL, Brown JP, Hellstrom I, Hellstrom KE, Carrasquillo JA and Larson SM (1984) High-level iodination of monoclonal antibody fragments for radiotherapy. J. Nuc. Med., 25, 367-70. Finn R, Balatoni J, Kothari P, Pentlow K, Sheh Y, Lom C, Dahl JR, Larson SM, Eckelman WC, Plascjak P and Adams HR (2001) Cyclotron production and potential clinical application of iodine-124 labeled radiotracers. In Application of Accelerators in Research and Industry, Duggan JL and Morgan IL (eds), American Institute of Physics, Woodbury, in press. Fraker PJ and Speck Jr, JC (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, l,3,4,6-tetrachloro-3a,6cc-diphenyl glycoluril. Biochem. Biophys. Res. Comm., 80, 849– 857. Francis GE, Mulligan W and Wormall A (1951) Labelling of proteins with iodine-131, sulphur-35 and phosphorus-32. Nature, 167, 748–751. Furniss BS, Hannaford AJ, Rogers V and Smith PWG (1978) Vogel's Textbook of Practical Organic Chemistry Including Quantitative Organic Analysis, 4th ed., Longman, New York, p. 599. Gabel CA and Shapiro BM (1978) 125I-diiodofluoresceine isothiocyanate: its synthesis and use as a reagent for labeling proteins and cells to high specific radioactivity. Analytical Biochemistry, 86, 396-406. Gilmore Jr, RC Robbins MC and Reid AF (1954) Labeling bovine and human albumin with I3I I. Nucleonics, 12, 65–68. Goodman MM, Callahan AP and Knapp Jr FF (1986) Design, synthesis and evaluation of 2-deoxy-2iodovinyl-branched carbohydrates as potential brain imaging agents. J. Labelled Comp. Radiopharm. 23,1269–1270. Goodman MM, Nefe KH, Ambrose KR and Knapp Jr., FF (1989) Effect of 3-methyl-branching on the myocardial retention of radioiodinated 19-iodo-18-nonadecenoic acid analogs. Nuc. Med. Bio., 16, 813-819. Goodman MM, Kabalka GW, Meng X, Daniel GB and Longford J (199la) Synthesis of iodine-123 labeled 4-0-(E)-3-iodopropen-2-yl-2-deoxy-D-glucose and 4-0-(E)-3-iodopropen-2-yl-D-glucose as potential brain and heart imaging agents. J. Labelled Comp. Radiophar., 30, 280-282. Goodman MM, Kabalka GW, Waterhouse RN and Daniel GB (1991 b) Synthesis of iodine-123 labeled 3-0(E)-3-iodopropen-2-yl-D-glucose: A potential new agent for the assessment of glucose transport into the brain and heart using SPECT. J. Labelled Comp. Radiopharm., 30, 278–79. Goodman MM, Kabalka GW, Meng X, Waterhouse RN, Knapp Jr. FF and Longford CPD (1992) Facile radioiodine incorporation via organometallics. In Synthesis and Application of Isotopically Labeled Comp. 1991, Buncel E and Kabalka GW (eds), Elsevier Science Publishers, Amsterdam, pp. 353-58. Hamill TG, Burns HD, and Gibson RE (2001 a) Radioiodinated a\-adrenergic receptor ligands, J. Labelled Compounds Radiopharm., 44, 61–72. Hamill TG, Duggan ME and Perkins JJ (2001b) The synthesis of the a,B3 integrin receptor ligand [125I]L775219. J. Labelled Comp. Radiopharm., 44, 55-60.

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Heideman Jr. ML, Levy RP, McGuire WL and Shipley RA (1965) 131I-labeled bovine thyrotropin: preparation through a new micro technique of iodine distillation and studies of absorption to glass and polyethylene. Endocrinology, 76, 828-838. Heindel ND (1978) Principles of target tissue localization of radiopharmaceuticals. In The Chemistry of Radiopharmaceotials, Heindel ND, Burns HD, Honda T and Brady LW (eds), Masson, New York, pp. 11–33. Helmkamp RW, Contreras MA, and Bale WF (1967) I 131 -labeling of proteins by the iodine monochloride method. Int'lJ. Appl. Rad. Isot., 18, 737-746. Holowka D (3981) N-chloro 125I-iodotyramine: an alkylating agent with high specific activity. Analytical Biochemistry, 111, 390–397. Hughes WL (1957) The chemistry of iodination. Annals of the New York Academy of Sciences, 70, 3-18. Hunter WM and Greenwood FC (1962) Preparation of iodine-131 labelled human growth hormones of high specific activity. Nature, 194, 495-496. Kabalka GW and Gooch EE, (1981) Syntheses of organic iodides via reaction of organoboranes with sodium iodide. J. Org. Chem., 46, 2582–2584. Kabalka GW and Varma RS (1989) Synthesis of radiolabeled compounds. Tetrahedron, 45, 6601–6621. Kabalka GW, Gooch EE, Hsu HC, Washburn LC, Sun TT, and Hayes RL (1982) Rapid and mild syntheses of radioiodinated estrogen derivatives via organoborane technology, App. Nuc, Radiochem., 197–203. Kemshead JT and Hopkins K (1993) Uses and limitations of monoclonal antibodies (MoAbs) in the treatment of malignant disease: a review. J. Royal Soc. Med., 86, 219-224. Knapp Jr, FF, Goodman MM, Callahan AP and Kirsch G (1986) Radioiodinated 15-(p-iodophenyl)-3,3dimethylpentadecanoic acid: a useful new agent to evaluate myocardial fatty acid uptake. J. Nuc. Med., 27, 521–531, Kocher DC (1981) Radioactive Decay Data Tables, Smith JS (ed), Technical Information Center, USDOE, Oak Ridge, pp. 121–134. Kohler G and Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 494–497. Koziorowski J (1998) Radiohalogenation of biomolecules an experimental study on radiohalogen preparation, precursor synthesis, radiolabeling and biodistribution. Acta Universitatis Upsaliensis, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 380, 57pp. Krohn KA and Welch MJ (1974) Studies of radioiodinated fibrogen-II, lactoperoxidase iodination of fibrinogen and model compounds. Int'l J, Applied Rad. Isot., 25, 315–323. Lambrecht RM and Wolf AP (1973) Cyclotron and short-lived halogen isotopes for radiopharmaceutical applications. In Radiopharmaceoticals Labelled Compounds, Vol. 1, IAEA, Vienna, pp. 275-290. Lambrecht RM, Mantescu C, Redvanly CS and Wolf AP (1972) Preparation of high purity carrier-free iodine-123 monochloride as a reagent for the synthesis of radiopharmaceuticals, IV. J. Nuc. Med., 13, 266-273. Larson SM, Carrasquillo JA and Reynolds JC (1984) Radioimmunodetection and radioimmunotherapy. Cancer Investigation, 2, 363–381.

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Mach JP, Carrel S, Ritschard J, Donath A and Alberto P (1980) Tumor localization of radiolabeled antibodies against carcinoembryonic antigen in patients with carcinoma: a critical evaluation. New Eng. J. Med., 303, 5-10. Maltly PJ (1994) Official and non official radiopharmaceuticals and their properties. In Textbook of Radiopharmacy, Sampson CB (ed), Gordon and Breach Science Publishers, Reading, pp. 193–200. Marchalonis JJ (1969) An enzymic method for the tracer iodination of immunoglobulins and other proteins. Biochemical Journal, 113, 299-305. Markwell MAK (1982) A new solid-state reagent to iodinate proteins. Analytical Biochemistry, 125, 427– 432. Mather SJ, (1994) Radiolabelled antibodies as radiopharmaceuticals. In Textbook of Radiopharmacy, Sampson CB(ed), Gordon and Breach Science Publishers, Reading, pp. 87–101. Matzku S, Kirchgessner H and Nissen M, (1987) Iodination of monoclonal IgG antibodies at a substoichiometric level: immunoreactivity changes related to the site of iodine incorporation. Nuc. Med. Bio., 14, 451–457. Mausner LF, Prach T and Richards P (1984) Production of radionuclides for generator systems. In Radionuclide Generators, Knapp FF and Butler TA (eds), American Chemical Society, Washington, DC, pp. 77–96. McFarlane AS (1956) Labelling of plasma proteins with radioactive iodine. Biochemical Journal, 62, 135– 143.

McFarlane AS (1958) Efficient trace-labeling of proteins with iodine. Nature, 182,53. Miyachi Y, Vaitukaitis JL, Nieschlag E and Lipset MB (1972) Enzymatic radioiodination of gonadotropins. J. Clinical Endo, Metab., 34, 23–28. Myers WG, Anger HO, Lamb JF and Winchell HS (1973) 123I for applications in diagnosis. In Radiopharmaceotical Labelled Compounds, Vol I, International Atomic Energy Agency, Vienna, pp. 249-256. Nikula T (1998) Development of radiolabeled monoclonal antibody constructs: capable of transporting high radiation dose into cancer cells. Biological Research Reports from the University of Jyvaskyla, 67, pp. 17-27. Nikula TK, Bocchia M, Curcio MJ, Sgouros G, Ma Y, Finn RD and Scheinberg DA (1995) Impact of the high tyrosine fraction in complementary determining regions: measured and predicted effects of radioiodination on IgG immunoreactivity. Molecular Immunology, 32, 865-872. Nozaki T. (1983) Other cyclotron radionuclides. In Radionuclides Production Vol II, Helus F (ed), Chemical Rubber Company, Cleveland, pp. 114–117. Owunwanne A, Patel M and Sadek S (1995) Iodine radiopharmaceuticals. In The Handbook of Radiopharmaceuticals, Chapman and Hall Medical, London, pp. 106–117. Ponto JA, Chilton HM and Watson Jr NE (1990) radiopharmaceuticals for genitourinary imaging: glomerular and tubular function, anatomy, urodynamics and testicular imaging. In Pharmaceuticals in Medical Imaging, Swanson DP, Chilton HM and Thrall JH (eds), MacMillan Publishing Company, New York, pp. 501–536. Pressman D and Keighley G (1948)The zone of activity of antibodies as determined by the use of radioactive tracers; the zone of activity of nephritoxic antikidney serum. Journal of Immunology, 59, 141–146.

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Pressman D and Korngold L (1953) The in vivo localization of anti-Wagner-osteogenic sarcoma antibodies, Cancer, 6, 619–623. Qaim SM and Stocklin G (1983) Production of some medically important short-lived neutron-deficient radioisotopes of halogens. Radlochimica Acta, 34, 25–40. Ramachandran LK (1956) Protein iodine interaction. Chemical Reviews, 56, 199-218. Rao MDP and Padmanabha J (1981) Kinetics and mechanism of iodination of phenol and substituted phenols by iodine monochloride in aqueous acetic acid. Ind. J. Chem., Section A, 20, 133–135. Redshaw MR and Lynch SS (1974) An improved method for the preparation of iodinated antigens for radioimmunoassay. Journal of Endocrinology, 60, 527-528. Rosa U, Scassellati GA and Pennisi F (1964) Labelling of human fibrinogen with 131I by electrolytic iodination, Biochimica et Biophysica Acta, 86, 519–526. Schmeisser M (1963) Handbook of Preparative Inorganic Chemistry, Vol I, 2nd ed. Brauer G (ed), Academic Press, New York, pp. 277-333. Seevers RH and Counsell RE (1982) Radioiodination techniques for small organic molecules. Chemical Review, 82, 575–590. Stadie WC, Haugaard N and Vaughan M (1952) Studies of insulin binding with isotopically labeled insulin. J, Bio. Chem., 199, 729–739. Stockiin G (1977) Bromine -77 and iodine-123 radiopharmaceuticals. In Radiopharmaceuticals and Other Compounds Labelled with Short-Lived Radionuclides, Welch M (ed), Pergamon Press, Oxford, pp. 131-148. Thorell JI and Johansson BG (1971) Enzymic iodination of polypeptides with iodine-125 to high specific activity. Biochimica et Biophysica Acta, 251, 363–369. Thrall J (1990) Radiopharmaceuticals for endocrine imaging. In Pharmaceuticals in Medical Imaging, Swanson D, Chilton H and Thrall J (eds), MacMillan Publishing Company, New York, pp. 343-393. Tjuvajev JG, Finn RD, Kyoichu W, Revathi J, Takamitsu O, Kennedy J, Beattie B, Koutcher J, Larson S, and Blasberg R (1996) Non-invasive imaging of herpes virus thymidine kinase gene transfer and expression: a potential method for monitoring clinical gene therapy. Cancer Research, 56, 40874095. Vaidyanathan G, Affleck DJ and Zalutsky MR (1997) Method for radioiodination of proteins using Nsuccinimidyl 3-hydroxy-4-iodobenzoate. Bioconjugate Chemistry, 8,724-729. Van den Abbeele AD, Aaronson RA, Daher S, Taube RA, Adelstein SJ and Kassis AI (1991) Antigenbinding site protection during radiolabeling leads to a higher immunoreactive fraction, J. Nuc. Med., 32,116–122. Van Dort ME and Hagen CA (2001) Synthesis of (E)-4-[4,4-dimethyl-2,5-dioxo-3-{l'-(125I)iodo-l'-propen3'-yl)-l-imidazolidinyl]-2-trifluoromethylbenzonitrile: a potential radioligand for the androgen receptor. J. Labelled Comp. Radiopharm., 44, 47-54. Wagner Jr. HN (1996) The second 50 years. In Nuclear Medicine 100 years in the Making, Wagner Jr. HN and Seaborg GT (eds), Society of Nuclear Medicine, Reston, pp 7–19. Waldman RA (1991) Monoclonal antibodies in diagnosis and therapy. Science, 252, 1657–1662. Welch MJ (1970) Labeling with iodine-123. The reactivity of iodine-123 formed by the decay of xenon-123. J. Am. Chem. Soc., 92, 408-409.

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14. RADIOBROMINE FOR IMAGING AND THERAPY DOUGLAS J. ROWLAND, TIMOTHY J. MCCARTHY AND MICHAEL J. WFXCH

Washington University in St. Louis, Mallinckrodt Institute of Radiology,510 S. Kingshighway Boulevard, Campus Box 8225, St. Louis, MO 63110, U.S.A.

INTRODUCTION The emphasis on radiobromine isotopes as imaging agents has shifted over the past 25 years from a concentration on 77Br and 82Br to 76Br. This shift is primarily due to the fact that PET cameras have gained importance in medical centers owing to their increased resolution capabilities over SPECT cameras. The bromine isotopes in use contain a broad range of practical half-lives, of which two are positron emitters worth utilizing as PET imaging agents. The other isotopes have significant electron capture or f}~, these decay properties may make them more useful as therapeutic agents than imaging agents. For example, although 77Br is no longer discussed in terms of its imaging capabilities as a SPECT agent, it has been suggested as a possible therapeutic isotope (ODonoghue & Wheldon 1996). The current status of the use of bromine in nuclear medicine research is the subject of this chapter PROPERTIES OF BROMINE ISOTOPES From an imaging standpoint, the ideal isotope should have a low energy positron emission with no electron capture, few background gamma rays, and a high specific activity. For therapeutic purposes, an isotope should decay mainly by electron capture and deposit all of its decay energy in a localized area. From a radiochemical perspective, bromine has several useful isotopes that fit both the criteria for imaging and therapeutic purposes, whereas fluorine has one isotope that is useful only for imaging. Bromine isotopes have half-lives ranging from 18 minutes for 80Br to 57 hours for 77Br Table 1. However, only two of these isotopes are practical as PET imaging agents, with a much narrower range in their half-lives. These are 75,76Br with 1.6 and 16.2 hour half-lives respectively. This pronounced difference in half-life can be the basis for selection of the isotope appropriate to the application. For faster physiologic processes 75Br is more suited for imaging, while 76Br can be used to study longer time frame physiologic processes, such as slowly proliferating tumors. Overall 75Br has better physical properties than 76Br for PET imaging, since 76Br has a high positron energy and many background gamma rays. However, the production method for 76Br is more practical for medical institutions, see the section on isotope production. Also, 75Br decays to 75Se, which has a long half-life of 120 days, which may not be feasible because of dostmetry issues. The other bromine isotopes while not useful for PET imaging might be useful as therapeutic agents in cancer treatments. The bromine isotopes that have a significant decay via electron capture have associated Auger electrons. When bromine decays by this route, an inner shell electron is captured by the nucleus leaving a vacancy in the inner atomic orbital. As the orbitals are reconfigured from this excited state to a ground state electrons can Handbook of Radiopharmaceuticals. Edited by M. J, Welch and C, S. Redvanly. ©2003 John Wiley & Sons, Ltd

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be emitted as Auger electrons. Kassis et al. have estimated that the average emission should be "several low energy electrons (20-80eV) with extremely short ranges (10-50A) in biological matter" (Kassis et al. 1982). Their assumption was that if the radioisotope was incorporated into DNA the proximity of the energy density from the decay could damage DNA beyond repair. Therefore radiopharmaceuticals have focused on compounds that are incorporated or come in close contact to DNA (77BrUdR, a thymidine analog (Kassis et al. 1982), and receptor based molecules such as steroids (DeSombre et al. 1988; DeSombre et al. 1996; Yasui et al. 2001)).

Table 1. Selected properties of the most commonly used bromine isotopes. Specific activity is given as the maximum theoretical specific activity.

Isotope 75

Br

T1/2 96.7 min

Decay Mode ^ ,. „ . (% Branching Ratio) p+ (73.02)

Specific Activity (mCi/ujnol)

P^ (MeV)

1944

2.008

Main v Pmorn

(MeV)

0.719

EC (26.98) 76

Br

16.2hr

P+ (54.7)

(% Abundance) 0.286(88) 0.511 (146.04)

193

3.941

1.180

EC (45.3)

0.511(109.46) 0.559 (74.0) 0.657(15.9) 1.216(8.8) 1.854(14.7)

2.951 (7.4) 77

Br

57.036 hr

P+ (0.74)

54

0.343

0.152

EC (99.26)

0.239(23.1) 0.297(4.16) 0.511 (1.48) 0.521 (22.4)

80

Br

80mBr 82

Br

17.68 min

10635

4.4205 hr

P'(91.7) p+ (2.2) EC (6.1) IT (100)

35.30 hr

P'(IOO)

889

0.781

2.001 0.849

0.368

0.142

2.316

709

0.511(4.40) 0.616(6.7) 0.037(39.1) 0.554(70.8) 0.619(43.4) 0.698 (28.5) 0.777 (83.5) 0.828 (24.0) 1.044(27.2) 1.317(26.5) 1.475(16.32)

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COMPARISON OF BROMINE TO FLUORINE AND IODINE When determining the efficacy of radiobromine as a biological diagnostic and/or therapeutic tracer, its comparison must be made to other radiohalogens. The two most commonly used halogens are fluorine and iodine, specifically the isotopes 18F, 1231,124I, and 125I. The chemistry of bromine is similar to that of these two elements, however its chemical properties lie between the two, Table 2. The reactivity of bromine is greater than iodine but is milder than fluorine. While the binding strength of the Br-C bond is only half that of the F-C bond, it is stronger than the I-C bond. This lends greater stability to brominated compounds in vivo, i.e. less dehalogenation occurs than with iodine. Therefore, from a chemical standpoint bromine has some attractive properties when compared to fluorine and iodine. Table 2. Chemical properties of the halogens. Element F Br 1

Electronegativity 4.1 2.7 2.2

Van der Waals Radius (A)

1.35 1.95 2.15

Binding Strength (kcal/mol) 114 59 45

A radiochemical comparison of bromide to iodide in vivo reveals an advantage. After in vivo metabolism a large exposure to the thyroid is not of concern. Unlike iodide, which traffics to the thyroid, bromide does not accumulate there. The biodistribution of bromide has been studied extensively in mice and rats (Cole & Patrick 1958; Rauws 1975; Rauws & Van Logten 1975; Soremark & Ullberg 1960). This information is important since brominated compounds metabolize in vivo to bromide. Depending on the rate of metabolism to bromide the background radiation from free bromide may render imaging agents ineffective and cause therapeutic agents to become harmful to non-target areas of the body from a dosimetric point of view. Bromide distributes rapidly, yielding close to its final distribution after only five minutes (Soremark & Ullberg 1960). Soremark et al found that bromide blood levels remained high, were greater than that in other organs and the rate of bromide excretion was slow (Soremark & Ullberg 1960). Raws and Van Logten state that the biological half-life of bromide is 3.5 days for rodents (Rauws & Van Logten 1975). Further studies showed that the biological half-life of bromide in dogs and man was 25-46 and 9-12 days, respectively (Trepanier & Babish 1995; Vaiseman et al, 1986). Raws and Van Logten investigated how dietary chloride affects the excretion rate of bromide (Rauws & Van Logten 1975). It was found that with a low salt diet the half-life increased to 25.1 days. As they increased salt intake they were able to lower the half-life to 2.5 days. This demonstrates why it is important to understand the excretion of bromide when using radiobrominated Pharmaceuticals since metabolism to bromide may affect the biodistribution of the radioactivity seen. If this is expected it may be possible to mathematically correct for the distribution of non-specific radioactivity, as was done by Gardell et al. (Gardelle et al. 2001). (Ribeiro et al. 1999) performed PET imaging with Br and compared it directly to F. The object of this study was to compare the quantification of small structures with PET. They found greater blurring of the images when using 76Br than with !8F. They concluded that since the intrinsic resolution of scanners has fallen from 8-12 mm to 2-5 mm, the effects of positron range on image quality must be re-evaluated. They further noted that if images

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from different isotopes are to be compared, corrections for the positron range may have to be made. PRODUCTION OF RADIOBROMINE ISOTOPES There are two methods of production for the various bromine radioisotopes. These are the indirect and direct methods of production. The indirect method utilizes the production of a parent nuciide that will decay to a radioactive daughter atom of the isotope of interest. The direct method involves producing the radionuclide of interest without first producing a parent nuclide. The use of these methods in the production of the isotopes in Table 1 will be discussed in the following sections. The productions themselves take place either at nuclear reactors to make use of the neutron flux or at accelerators making use of p, d, 3He, or a particles to induce nuclear transformations to the isotopes of interest. 80,80m,82

Br ISOTOPES

8o,80m,82gr {g^p^ are produced by irradiating natural bromine in a reactor neutron flux via the reaction *Br(n,y)x+1Br (Wong & Ache 1976). Since these radioactive bromine isotopes are produced from non-radioactive bromine isotopes and the two cannot be separated from each other chemically, they are not obtainable in high specific activity as would be needed for receptor and therapeutic studies. Both 80Se and 82Se are stable nuclides, therefore it may be interesting to study the production of the three bromine isotopes with the 80Se(p,n) and 82 Se(p,n) reaction at a cyclotron, which would provide higher specific activities. However, this is only interesting from the standpoint of using them as therapeutic agents, since their properties are not compatible with PET imaging. 75

Br Both direct and indirect methods for producing 75Br have been suggested. The indirect reaction utilizes the production of 75Kr via na'Br(d,xn), followed by its decay to 75Br with a half-life of 4.3 minutes (Qaim & Weinreich 1981). There are several direct methods for the production of 75Br. The first is a (p,a) reaction on 78 Kr, this reaction requires the lowest beam energy, 15->12MeV (Friedman et al. 1982). Both this reaction and the indirect method have low yields and are therefore of little interest for routine production. In addition in the production of 75Kr contamination with 77Br is unacceptably high at 7.7%. In order to produce large quantities of 75 Br, the targets must use isotopically enriched arsenic or selenium. Both of these target materials have lower than ideal melting points and thermal conductivity to allow for high beam current irradiation. In order to account for this, copper alloys with this metal have been used to increase their melting points. With arsenic the preferred reaction is 75As(3He,3n)75Br with a Cu375As alloy, (Weinreich et al. 1980), giving a 8.1 mCi/uA-h production rate. This requires a higher beam energy compared to the other reactions, 36-^25MeV. The main contaminant in this reaction is 76Br (2%). The reaction 76Se(p,2n)75Br using both 76Se (Paans et al. 1980) and Cu,76Se (Maziere et al. 1984) targets at 28->22MeV have been investigated and show 76Br as the major contaminant at the 1.4% level. The reaction using the Cu2Se alloy has 3-fold lower rate of production than the solid Se target. 119mCi/fiA-h compared to 43mCi/uA-h. Reactions on 76Se targets at a lower energy have been investigated at 24->21.5MeV (Maziere & Loch 1986). However, the production rate is higher, about three times, and the contamination is lower, about half, at the higher energy making it more useful than at lower energy. 76

Br As with 75Br, both direct and indirect methods of production have been used for the production of 76Br. The

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indirect method utilizes the decay of 76Kr with a 14.6 hour half-life (De Jong et al. 1979; Qaim 1986; Qaim et al. 1977; Sargent et al. 1975) using various reactions, (p,xn), (d,xn) and (3He,3n), at high energies (SOMeV deuterons and 65MeV protons). These reactions have low rates ranging from 0.081 to 0.459mCi/pA-h and have a large 77Br contamination. The direct methods use the (3He,2n) reaction on 75As and Cu375As at 30MeV and 18->10MeV, respectively, with low contamination from 77Br (Maziere et al. 1984; Qaim 1986). These reactions have moderate yields at 0.703 and 0.297mCi/|iA-h. More recently, the direct reaction of Cu276Se alloy with protons at 16MeV has become an important production method (Tolmachev et al. 1998). This group achieved up to 1.9mCi/jxA-h with less than 2% 77Br contamination from trace amounts in the enriched 76Se. This rate of production is very good and can be performed on small medical cyclotrons and therefore can be taken advantage of by many institutions. 77

Br The production of 77Br can be achieved with a variety of nuclear reactions via the indirect and direct methods. The indirect methods tend to have higher yields of the parent nuclide 77Kr with a 0.5 hour half-life. The total yield of 77Br via this method is limited to a few mCi because of the short half life of the parent (Maziere & Loch 1986). Arsenic and selenium are again the materials of choice to produce 77Br in direct reactions, although natMo is used in spallation production. The spallation production natMo(p,spallation)77Br occurs at 800MeV and has a large production rate compared to the other reactions (Grant et al. 1981). The target for this method is irradiated for 3-4 days, in a parasitic position. The main contaminate is 76Br at the 2% level. However in commercial productions up to several hundred mCi can be obtained using this method. For other production methods on small medical cyclotrons the yields are typically much smaller. The (a,2n) reaction is the most common production using75As at energies between 14 and 30MeV (Blessing & Qaim 1984; Blessing et al. 1982; Helus 1970; Nunn & Waters 1975). With this method 50mCi has been produced in a single batch. The main contamination again comes from 76 Br (5%). This isotope may also be produced when 77Se is used at a lower energy (12MeV) with 2.5% 76Br contamination due mainly to the target enrichment (Norton et al. 1978). The production rate is moderate at 0.51mCi/uA-h in this reaction but at this energy it is accessible to small medical cyclotrons. SEPARATION METHODS There are a variety of production methods for the different bromine isotopes and for each production method a separate purification procedure has been elaborated. The two methods that have been utilized are the wet chemical and the dry distillation. The wet chemical separation has been principally used for the preparation of 77 Br from spallation products in irradiated Mo foils (Grant et al. 1981). With the growing interest in using 76Br and 77Br for PET imaging and therapy, production methods have focused more recently on the more practical CujSe targets that can be reused with little maintenance. The isotopes are separated from these reusable targets via the dry distillation technique. Only this latter method will be discussed in detail here. DRY DISTILLATION The dry distillation method of Tolmachev et al. (Tolmachev et al. 1998) is the most recent method described in the literature for the production of 76Br on a low energy cyclotron. Their distillation method is based upon work performed by (Janssen et al. 1980 263; Kovacs et al. 1985; Vaalburg et al. 1985). The dry distillation technique is a thermal chromatographic method. The irradiated target is heated in a furnace to just below its melting point (approximately 1100°C) in a quartz tube that is continually flushed with argon gas. A polytetrafluoroethylene

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(PTFE) tube is inserted into the quartz tube outside the main furnace and the other end is inserted into a dilute NaOH solution to trap any excess bromide in the argon carrier gas. When the target is heated, Se and Br are volatilized and carried through the system by the argon gas. The bromide condenses onto a small section of the PTFE tubing separately from the Se. The section of tubing with the deposited bromide is excised and washed with approximately 1 ml of ultra-pure water or absolute ethanol. At this point the activity is ready for use in labeling of compounds. LABELING METHODS A variety of labeling methods have been employed to radiobrominate compounds. The reactions are divided into three main classes dependent on the properties of the physical process that takes place. These are enzymatic, chemical and decay-induced reactions.

DECAY INDUCED LABELING This method employs the decay of a parent isotope into a reactive daughter species. It has been used for bromine isotopes by utilizing the decay of 77Kr via $* and electron capture decay or 76Kr via electron capture decay. The P+ decay of 77Kr gives rise to negatively charged 77Br which is then available for reaction. The electron capture decay of 76,77Kr yields multi-positively charged bromine species due to the emission of Auger electrons. The final decay induced labeling reaction uses the isomeric transition of 80mBr to the ground state that is followed by an Auger electron cascade. Once the reactive bromine species is generated and is in the presence of substrate it can undergo one of the chemical labeling reactions. This method is good for rapid bromination in a carrier free environment. However, there are drawbacks to this type of reaction. Typically the yields are low and vary quite a bit from reaction to reaction. There is also low specificity to a single reaction site due to the decay-daughter being highly energetic and reactive. The decay-induced is probably the least common method due to its low yields. Also with the use of compact medical cyclotrons and reusable targets that give large quantities of carrier free radiobromine directly, it is not a necessary labeling method.

CHEMICAL LABELING Electrophilic Electrophilic reactions occur via one of three routes. These are the oxidation of molecular bromine, heterogenous bromine compounds, and demetallation reactions. Maziere and Loc'h thoroughly reviewed these reactions previously (Maziere & Loc'h 1986). Only brief overview of the chemistry will therefore be given here for these reactions. The oxidation of molecular bromine is performed by the addition of manganese oxides (MnO2 and MnO 4 ) in an acidic medium followed by a distillation or extraction step. Compounds that have been synthesized via this reaction are dimethoxyphenylisopropylamine (a psychoactive compound) (Sargent et al. 1975), spiperone (Kulmala et al. 1981) and ergocryptine (Markey et al. 1976) (dopaminergic compounds), estrogen derivatives46 and fatty acids (Machulla et al. 1980). The heterogenous bromine compounds use an oxidizing agent to increase their electrophilic reactivity towards aromatic compounds. The oxidants most commonly used are H2O2, imides and amides. As an example,

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hypobromous acid is generated in situ via a reaction with peracetic acid: CH3CO2H + H2O2 •> CH3CO3H : CH3CO3H + Br -> HOBr With this reaction, synthetic yields have been found to be in the 70-90% range with specific activities in the range of 1 -2 Ci/UMnol (Maziere & Loch 1986). The reaction time was found to be short, allowing for labeling with the short half-life 75Br. This technique has been used to label receptor-binding compounds such as hormones or dopaminergic antagonists. ,N-Chloro compounds have been employed most often as the oxidizing agents. The most commonly used compounds are N-chlorotoluenesulfonamide (chloramine-T and dichloromine-T) and Nchlorosuccinimide in one-pot synthesis. The reaction conditions using the N-chloro compounds are milder than the hypohalites. However, with the presence of chlorine in the oxidant, chlorination of the substrate can be a significant side reaction. The chlorination reactions do, however, proceed at a much slower rate than the main reaction; therefore if the reaction time is reduced the chlorination by-product will be minimized. Co-chlorination of substrate was investigated in detail for chloramine-T (one of the most widely used oxidants) by (Suehiro et al. 1990). The electrophilic demetallation reaction proceeds through the same mechanism as the former reactions except bond cleavage is at a C-metal bond and not a C-H bond on the substrate molecule. The C-metal bond is weak and has a higher bond polarity than the C-H bond, thus making it more susceptible to attack by an electrophile. Metal species that have been investigated for radiohalogenation since 1980 are organometallics containing mercury, thallium, boron, tin, silicon, germanium and lead, see (Adam 1986) for a complete review of these demetallation reactions. There are several other advantages to using this type of reaction over those previously discussed (Coenen et al. 1983). A one-pot synthesis can take place in which reaction times are shorter and labeling conditions are milder due to the lability of the metal derivatives. Better yields are obtained due to the higher sensitivity of the C-metal bond to electrophilic attack. Also the bromination is directed regiospecifically because of the polarity of the C-metal bond. Since metallic impurities can be toxic, the separation of the compound of interest from metallic species must be validated. The US pharmocopeia gives tests for determining if the level of metallic impurities is under allowable limits (USP/NF 2001). Nucleophilic Displacement Nucleophilic reactions can be broken down into two types of reactions, the exchange of bromine and an organic substituent (SN2) or bromination via diazotisation (SN1). The advantage of these reactions are the simplicity and the fact that radiobromine is usually separated from the target as bromide. Therefore no oxidant need be added to the reaction mixture. A radiochemical yield of 10-20% was obtained for the preparation of 3-deoxy-3-bromo~Dglucose by the substitution of bromide on the triflate derivative of the sugar (Kloster et al. 1983). Fatty acids have also been synthesized in 36-53% yields using tetrachloromethane and triphenylphosphine as reagents, however the chloro compound needed to be separated from the radiobromo compound (Kilbourn et al. 1982). Aside from these reagent systems, interhalogen exchanges have been utilized where a Br-for-I exchange takes place. Radiobromo fatty acids have mainly been produced via this method (Machulla et al. 1978) and norcholesterol analogs have also been studied (Kojima et al. 1977a; Kojima et al. 1977b). One drawback to these reactions is that except for alkyl bromides aromatic compounds cannot be synthesized by this method (Huang & Friedman 1983). (Moerlein et al. 1988) addressed this disadvantage via a cuprous chloride-assisted-nucleophilie

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bromodeiodination of bromospiperone. Substitution onto phenyl rings can be performed via diazo derivatives of aromatic compounds. This is a Sandmeyer type reaction that allows halogenation of an electronically deactivated or non-activated ring in a predetermined position. Several other modifications of the classic reaction have been described and were reviewed by Maziere & Loch (1986). ENZYMATIC LABELLING There are a variety of phenol containing compounds that can be labeled by enzymatic catalysts. The commonly used enzymes are chloroperoxidase (Knight et al. 1977), bromoperoxidase (McElvany & Welch 1980) and myeloperoxidase (McElvany et al. 1980). They have continued to be used since the early eighties. Labeling is rapid and proceeds under mild conditions in a no-carrier added environment. Labeling of proteins is especially suited for these reaction conditions since proteins are prone to degradation under unfavorable conditions. The reaction takes place with the addition of the radiobromide and H2O2 to the substrate in the presence of the enzyme. The enzymatic reaction with chloroperoxidase works best at a pH of 2.8. Yields of 20-80% have been achieved with subtrates such as albumin, thyroglobulin, tyrosine, DOPA, serotonin, uracil, and cytosine (Friedman et al. 1979; Hadi et al. 1979; Knight et al. 1975). In order to avoid subjecting proteins to such a high pH, Knight et al. (1977) used an acylating agent (N-succinimidyl-3-(4-hydroxyphenyl)propionate) as the substrate at pH 2.8 with chloroperoxidase. The brominated compound was then purified and allowed to conjugate to fibrinogen or serum albumin at pH 8. A review of other enzymatic reactions can be found in Huang and Friedman (1983) and Maziere and Loch (1986). BROMINE RADIOPHARMACEUTICALS AND THEIR BIOLOGICAL UTILIZATION Bromine radiopharmaceuticals have been synthesized for a wide range of uses in the study of animal and human biology. The radiopharmaceuticals will be discussed in terms of their biology below. One point of information must be discussed at this point. In order to properly study the biological activity of these molecules it is important to determine their specific activity. The typical method for determining this quantity is to analyze a radioactive sample with HPLC (Kloster & Laufer 1983). Ultraviolet (UV) light detection can determine the total amount of compound, both cold and hot, and the amount of radioactive compound can be determined with a radioactive detector. However, compounds with a low absorption in the UV might not have enough sensitivity for a precise quantitative determination. Work is being done to circumvent this problem by measuring the ratio of 76Br to 79Br labeled compound with the use of Electrospray mass spectrometry. Forngren et al. (2000) claim to have improved the sensitivity and selectivity [separation of mass-to-charge ratio (m/z)] in specific activity measurements. MISCELLANEOUS Bromide Bromide is the simplest form in which radioactive bromine has been used. It is used for the estimation of extracellular space and blood volume. Miholic used [82Br]NaBr for these types of measurements in comparison to bioelectric impedance (Miholic et al. 1992). They attempted to validate bioelectrical impedance measurements with bromide (the gold standard) for the estimation of extracellular space and blood volume. The impedance measurements had a deviation as high as 20%, which would limit clinical use.

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Extracellular space volume was estimated from the ratios of bromide to chloride in urine and saliva (Morkeberg et al. 1992). Piglets were injected with 82Br and 36C1 and at 4 hr post injection, samples of plasma and urine were compared. Urine samples were used to determine extracellular water volume in adult and young animals. This required one sample before injection and one at 4-8 hr post injection. Cl concentration was measured and a correction factor describing the relationship of the ratio of Br to Cl in urine to the ratio in plasma was determined. Measurements in humans were valid for urine samples but error estimates were too large to make saliva measurements useful. 82

Br was used to study the channel opening of the GABAA receptor when exposed to chlordiazepoxide (a

tranquilizer) by looking at the transmembrane flux of the halide (Cash et al. 1995). Thrombus Detection The use of [77Br]fibrinogen for detection of deep vein thrombosis was described in a review by Krohn and Knight (1977). Fibrinogen is a large molecular weight plasma protein that is incorporated during thrombus formation and lysis. The fibrinogen uptake test takes place over days or weeks and therefore an isotope with a sufficiently long half-life was of necessary. Labeling of fibrinogen with 77Br takes place via a two-step procedure: (i) bromination of an acylating agent (N-succinimidyl-3-(4-hydroxy-phenyl)propionate (SHPP) and (ii) the conjugation of 77BrSHPP to fibrinogen. Alternatively fibrinogen can be synthesized via the following reaction: (i) carrier free chloroperoxidase bromination and extraction into benzene, (ii) drying and dissolution in buffer with fibrinogen in an ice bath. For these two methods yields were found to be 85% and 35-50%, respectively. Fatty Acids The use of bromine in labeling fatty acids has also been studied. In Huang and Friedman's (1983) review on the use of bromine, fatty acid use in nuclear medicine was presented. 77Br-a-bromostearic acid and 77Br-17bromoheptadecanoic acid were used to investigate myocardial metabolism. Abbas et al. (1991) looked at 2-(6-(4bromophenoxy)hexyl)oxirane-2-carboxylic acid for the detection and characterization of cardiornyopathies. The synthesis of the latter utilized a nucleophilic exchange reaction in acetic acid using Cu(I)Cl as catalyst with a 45% yield, 5-Bromo-2-Thiouraeil [76Br]5-Bromo-2-thiouracil was used to investigate human and murine melanotic melanoma with PET in rats (Mars et al. 2000). The preparation of the radiotracer was performed via an electrophillic reaction using Chloramine-T in phosphate buffer with thiouracil. Images were obtained 1 -day post injection of radiotracer for the detection of subcutaneous tumors. Images clearly showed that the radiotracer was retained in tumors as the radioactivity in the rest of the body declined. This study demonstrated the diagnostic potential of this agent, including dosimetry, prior to targeted radiotherapy.

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THYMIDINE ANALOGS 5-Bromo-2'-Deoxyuridine (BrlJdR or BrdU in the literature) BrUdR was radiolabeled as early as the 1960's. However, a simpler preparation of BrUdR from 5trimethylstannyl-2'-deoxyuridine (TMSUdR) was described by Koziorowski and Weinreich (1997). In this synthesis, l0uL of 76Br in 0.1 M sodium chloride/0.1M phosphate buffer at 7 pH was added to l0jiL aqueous TMSUdR (1 mg/ml) followed by the addition of 10uL of chloramine-T (2mg/ml in 0.1M pH7 phosphate buffer). Reaction time was 1 min with >90% yield. This reliable production with short labeling time decreases radiolysis and reduces personnel dose. As described earlier, bromide has a slow clearance and a broad distribution throughout the body, an understanding of the catabolism of compounds must be known in order for radioactive distributions to be understood in terms of their parent compound and of the catabolites. Kriss et al. (1963) investigated the in vivo stability of BrUdR in man and found after IV injection there was a rapid clearance from the blood,"...in part owing to its degradation associated with the liberation of bromide "(Kriss et al. 1963). At 1 hr post injection, it was found that 60% of the 82 Br activity was in intracellular or intralumenal sites. Practical usefulness of BrUdR hinges on the development of methods for reducing the relative amount of bromide released into the blood or for rapidly clearing the body of free bromide. Lu et al. (1999) studied the elimination of nonspecific radioactivity of bromide derived from the metabolism of BrUdR. Torasemide and NaCl were used as diuretics to enhance the elimination of free bromide. By doing this, the group found a 30-50% decrease in the radioactivity in the body with the highest uptake of radioactivity in organs with the highest DNA synthesis. The conclusion was that with the use of diuretics, the tracer could be useful for observing DNA synthesis with PET. Kassis et al. (1982) studied the lethality of Auger electrons from the decay of 77Br in BrUdR. They studied the effectiveness of the Auger electrons to kill V79 cells in vitro. Auger electrons have a high-linear energy transfer within their immediate surroundings that allows them to damage the cell. The radiotoxicity seen in this study was attributed to a deposition of l00eV in an approximately 10A sphere around the decay site in the DNA. They conclude that "the quality of the electron spectrum, site of intracellular localization and energy deposited in the nucleus and its radiosensitive regions" are needed for appropriate biological toxicity. They go on to suggest the possibility of using this compound for radiotherapy of tumors. [76Br]BrUdR was characterized for examination of proliferation potential in multicellular tumor aggregates in healthy rats and pigs (Bergstrom et al. 1998). In cell aggregates 30-90% of activity was recovered in the DNA fraction, with variable recovery dependent upon organ type. Their assessment was that corrections for free bromide were necessary in PET imaging or that it would be necessary to eliminate a fraction of the free bromide through forced diuresis. A comparison of [76,77Br]BrUdR to [125,131I]IUdR for in vitro and in vivo experiments in mice has been described (Ryser et al. 1999). This study showed a significantly higher incorporation of the brominated compound due to its more appropriate size, hydrophilicity and similarity to thymidine than the iodinated compound. However, both

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dehalogenated quickly in vivo but not in vitro. The final conclusion was that 76BrUdR could not considered more useful than 124IUdR from these experiments due to small differences in their characteristics and the difficulty in extrapolating their performance in mice to men. Boni et al. (1999) assessed tumor cell proliferation with 76BrUdR in human patients with metastatic melanoma. In one patient unexpected metastases was found on BrUdR PET and became clinically evident 4 weeks later. They suspected the result was more an effect of increased circulation in rapidly proliferating metastases rather than the incorporation of BrUdR into DNA. They suggest a search for more stable compounds. Both Gardelle et al. (2001) and Gudjonssona et al. (2001) find 76BrUdR unsuitable for use in PET due to its high metabolism and the subsequent long biological half-life of free bromide released. The future of this compound for measuring cellular proliferation in vivo with PET is not favorable. 5-Bromo-2'-Fluoro-2'-Deoxyuridine (FBAU or BFU in the literature) A method for the synthesis of FBAU was described by Misra et al. (1986). Their synthesis consisted of an electrophilic bromine monochloride in a no-carrier added reaction. [82Br]NH4Br in glacial acetic acid was added to the precursor. N-chlorosuccinimide in acetic acid was then added and heated for 60 mins. The mixture was transferred and washed with ethanol. The solvent was evaporated in vacuo. Ammonium hydroxide in ethanol was added and heated for 25 mins. The solvents were removed and the product was isolated via preparative TLC with 86% yield. Mercer et al. (1989) investigated the biodistribution of [82Br]FBAU in BDF1 mice bearing Lewis lung tumors. They found the in vivo stability of this compound to be much better than the non-fluorinated analog (BrUdR). Also, FBAU cleared rapidly through the kidneys and residual activity after 4 hrs was believed to be free bromide and the distribution followed that for bromide. Tumor uptake was minimal. They believe that thymidine competes for the same binding site for transport of nucleosides across the cell membrane. The measured inhibition constants showed that this compound was a weak competitor for the nucleoside transporter when compared to physiological nucleosides. They also concluded that the chemical properties that make FBAU stable in vivo also make it a poor substrate for mammalian kinase enzymes and thus probably not a good imaging agent because of the low uptake in tissues. The validation of FBAU as a proliferation marker in PET imaging was revisited by Lu et al. (2000). The synthesis paralleled the simple synthesis of BrUdR. It used 5-trimethylstannyl-2'-fluoro-2'-deoxyuridine as the precursor with the reaction proceeding via an electrophilic reaction with chloramine-T. The radiochemical yield was 80%. In the imaging studies the greatest uptake was seen in the spleen and intestines. A study of the localization of the activity found that 90% of activity was in the DNA. 95% of the intact tracer was accounted for in the urine. They also looked at injections of cimetidine to help increase the uptake of tracer into the cells by prolonging the circulation of the tracer. This increased the uptake by 2-5 times depending on the amount of cimetidine administered. Following the injection of cimetidine the urine excretion dropped markedly. They state that FBAU compound has a good potential as an imaging agent. FBAU has also been studied for its use as a proliferation indicator in four lung cancer cell lines and one endocrine tumor cell line (Xing et al. 2000). 6-diazo-5-oxy-L-norleucine (DON) was being assessed for its potential as an

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antitumor agent against these cell lines. The uptake of FBAU in DON treated cell lines demonstrated a decrease in proliferation and they therefore conclude that [76Br]FBAU could be used in treatment evaluation. Borbath et al. (2002) showed that FBAU could be used as proliferation marker with the co-injection of cimetidine to increase the bioavailability of FBAU. Doing this increased the sensitivity for PET detection. The compound was compared to 2-[14C]thymidine incorporation and the labeling index assessed with cold BrUdR. After 6 hrs post-injection more than 95% of the FBAU was DNA-associated. The DNA labeling with [76Br]FBAU showed a good correlation with the incorporation of the 14C compound and the labeling index measure with cold BrUdR.

RECEPTOR SITE DIRECTED Androgen 2a-[77Br]Bromo-5a-dihydrotestosterone

has been synthesized to investigate imaging of the prostate via SPET

imaging (Ghanadian et al. 1977). This group studied this compound in both rat and human tissue. The studies in the rat showed an uptake of 0.5-0.8% ID/g of tissue. In contrast the human studies showed a 200 fold lower value. This agent was found to be inappropriate for scanning the prostate. More resent work by Eakins and Waters (1979) showed that the low amount of localization of the compound was due to "rapid and extensive debromination" of the molecule. Estrogen 16a-[77Br]Bromoestradiol-17a was described by McElvany et al. (1982a). In this work they compared the brominated compound to the [125I]iodinated compound. Dosimetry and preliminary clinical studies were published at the same time (McElvany et al. 1982b). The iodinated and brominated compounds could be used interchangeably for the imaging of breast tumors containing estrogen receptors (ER). Preliminary clinical studies showed promise with the need for further development. The same group described in another study the synthesis of 16a-[77Br]Bromo-l ip-methoxyestradiol-l 7P with high specific activity (Katzenellenbogen et al. 1982). They found that it had a high affinity for the ER, a low nonspecific binding and was retained for prolonged periods. They suggested that it may be a better imaging agent for breast tumors than the ^a-j^BrJBromoestradiol-^a compound. 16a-[77Br]Bromoestradiol-17a and 16a-[77Br]Bromo-l lp-methoxyestradiol-173 were used to further investigate factors affecting target site uptake of estrogen radiopharmaceuticals (McElvany et al. 1983). There was a strong correlation on the uptake of the compounds with the stage of the estrous cycle. They stated that it is not only important to know the binding affinity of the agent for the ER, but knowledge of its interaction with non-receptor binding proteins is also necessary. In addition changes to endocrine and physiologic status affecting the uptake must be understood when using these compounds. Seevers et al. (1986) published a study on non-steroidal estrogens labeled with

80m

Br. ((E,Z)[l-(4-

dimethylaminoethoxy)phenyl]-l (4-hydroxyphenyl-2-bromo-2-phenylethylene and 1,1 -bis(p-hydroxyphenyl)-2bromo-2-phenylethylene were used as possible agents for the diagnosis and treatment of cancers. Both of these compounds were shown to bind strongly to the ER.

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DeSombre et al. (1988) also investigated the potential of 80mBr in estrogen directed therapy. They prepared 4Bromo-l,2-dihydro-l ,5-dimethyl-2-phenylpyrazol-3-one (bromoantipyrine) and 5-Bromo-2'-deoxyuridine to look at their therapeutic affects. In culture, the BrUdR was radiotoxic as shown in earlier work, causing chromosomal damage. However the bromoantipyrine was without effect. In 1990 this group compared steroidal and nonsteroidal bromovinylestrogens in ER positive cancers. The best tissue-to-blood ratios were found for E-17ct[77Br]bromovinyl-11p-methoxyestradiol, although it also had a high nonspecific uptake. 1,1-bis[4-hydroxy~ phenyl]-2-f77Br]bromo-2-phenylethylene had a comparable specific uptake at 2hr but showed better retention. (DeSombre et al. 1990). Norepinephrine [76Br]-meta-bromobenzylguanidine (MBBG), a functional analog of norepinephrine, was investigated for its potential for clinical use in heart disease (Loc'h et al. 1994; Maziere et al. 1995b). The synthesis uses an iodinated analog (MIBG) and [76Br]NH4 in a Cu+ assisted halogen exchange reaction which gives a 60–65% radiochemical yield. Metabolic analysis in rats showed a rapid breakdown of the compound whereas metabolism was slow in dogs. 85% of activity in heart after 25 hrs was intact compound in the rat studies. After 4 hrs the heart-to-lung concentration ratio was 8. This study showed that the myocardial uptake was similar to norepinephrine. They contend that this compound could be used in assessing heart catecholamine reuptake disorders with PET. MBBG was compared to MIBG for assessing imaging and targeting in xenografted PC12-pheochromacytoma tumors (Clerc et al. 1995). They concluded that MBBG efficiently targeted the tumors and is a promising compound for imaging and quantifying tumor uptake with PET. They continued investigating this compound for its kinetics in an isolated rat heart (Raffel et al. 1998). It was found that MBBG shares the same kinetic mechanism as norepinephrine and would be useful for assessing the sympathetic nerve status in the heart through PET imaging. Hallucinogens R(—)-2,5-Dimethoxy-4-77bromoamphetamine (77Br-R(—)DOB) was radiolabeled in high specific activity via a modified synthesis of Coenen (Coenen 1981; Wang et al. 1988). DOB is a stereospecific hallucinogen and the mechanism of action in man was unknown. The compound was used to label the 5-hydroxytryptamine receptor in the central nervous system of the rat. They believe this compound could be used to elucidate mechanism of hallucinogenic drug actions. The investigation of this compound was continued by studying binding affinities at nine neurotransmitter binding sites, LSD, N,N-dimethyltryptamine, and l-(2,5-dimethoxy-4-iodo-phenyl)-2aminopropane (Pierce & Peroutka 1989). These compounds were used to better comprehend the mechanism of hallucinogens in man. This was the first study using this compound in order to elucidate the DOB binding site in the human brain. Nicotink Acetylcholine Receptor The loss of neuronal nicotinic acetylcholine receptors (nAChRs) has been shown to occur in neurodegenerative disorders. The compound 5-[76Br]bromo-3-[[2(S)-azetidinyl]methoxy] pyridine ([76Br]BAP) was synthesized via an oxidative bromodestannylation reaction to investigated at this receptor (Sihver et al. 1999). The binding

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properties of this compound were studied in vitro and in vivo in the rat brain and via PET in two rhesus monkeys. They found that this compound is a high affinity ligand for the nAChRs and believe that it should have a low toxicity. They suggest it could be used in PET investigations with human subjects. Muscarinic-Cholinergic Receptors Both 4-[76Br]bromodexetimide (BrDEX) and 4-[76Br]bromolevetimide (BrLEV, the inactive enantiomer) were prepared to study their in vitro and in vivo characteristics with respect to the muscarinic-cholinergic receptors (mAChR) in the rat brain (Loch et al. 1996b). The compounds were prepared by electrophilic bromodesilylation using chloramine-T and no-carrier-added [76Br]NH4. BrDEX was selectively taken up in the cortex, striatum, thalamus and hippocampus while BrLEV showed no specific uptake. Further studies in primates showed selective accumulation in the cortex and striatum for BrDEX, while BrLEV showed similar uptake in the cerebellum compared to these other areas. BrDEX should be a good measure of the status of m-AChR and BrLEV is complementary so that non-specific binding of BrDEX can be measured. Dupont et al. (1999) used [76Br]BrDEX for in vivo imaging of humans with medial temporal lobe epilepsy. They showed a mismatch in the concentration of BrDEX in the temporal lobe of patients with this disease and conclude that the radiotracer could be used in studies of mAChR in humans. Three brominated quinuclidinyl benzilate (QNB) derivatives where synthesized via a destannylation reaction: (Z(,-)-[76Br]BrQNP, E(-,-)-[76Br]BrQNP, and E(-,+)-[76Br]BrQNP) (Strijckmans et al. 1997). The (-,-) configurations showed good specific uptake in mAChR rich structures of the brain and also in the heart with low metabolism rates. The (-,+) compound had a rapid clearance with low uptake and higher metabolism. The in vivo data suggested that the type of chiral center of a compound influences the affinity for certain subtypes of the mAChR. The (-,-) compound shows promise as a PET imaging agent in humans. A competitive study of

125

IQNP against 3-quinuclidinyl 2-(5-bromothienyl)-2-thienylglycolate (BrQNT)

demonstrated the in vivo selectivity of BrQNT for the M2 subtype receptor (Cohen et al. 1998). The regions that are enriched in the M2 subtype of the receptor preferentially block IQNP uptake. They conclude that BrQNT is selective for M2 in vivo and could be a potential ligand for use with PET imaging with [76Br]. Serotonin Transporter 5-[76Br]bromo-6-nitroquipazine was synthesized to study its efficacy in imaging the serotonin transporter in the brain (Lundkvist et al. 1999). This compound was prepared from N-t-BOC protected trimethylstannyl precursor via an electrophilic substitution of 76Br in the presence of chloramine-T. In rats they found that the compound enters the brain rapidly and paralled the well-known distribution of serotonin transporters in the midbrain, pons, thalamus, striatum and neocortex. Metabolism was rapid but greater than 90% of the activity in the brain represented unchanged compound. PET imaging in a baboon followed this study. They found uptake in the thalamus, striatum and pons (1.5 times the cerebellum at 3hr and 2.5-4 times higher at 24hr). They conclude that this compound could be developed for studying pathological and pharmacological variations in the density of serotonergic nerve terminals in the human brain with PET. Benzodiazepine Receptor The benzodiazepine receptor in the brain was investigated with NNC 13–8199 labeled with 76Br (Foged et al.

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1997). The compound was prepared via a destannylation reaction in the presence of chloramine-T. In monkeys the compound was taken up into the occipital, temporal and frontal cortex. Greater than 98% of the compound remained unchanged in the brain. The compound has a great potential for in vivo imaging in the human, Dopamine Receptors In the mid 1980's an intense effort was placed into mapping cerebral dopaminergic receptor sites in vivo. This effort was spurred by the involvement of dopamine pathways in neurological disorders. De Jesus and Friedman (1986) reviewed the state of p-bromospiroperidol (BrSP) with 75,76, or 77Br in 1986 and described the future directions of dopamine receptor studies using PET. Moerlein et al. used [77Br]BrSP and other butyrophenone neuroieptics to study the rat brain and image brains of the baboon (Moerlein et al. 1986; Moerlein & Stocklin 1984). The synthesis used no-carrier-added bromine mixed with benperidol in trifluoroacetic acid with dichloramine-T. They found that the compound had a larger uptake in the striatum as compared to the cerebellum, cortex and blood. The work on dopaminergic receptors continued by studying the effect of lipophilicity on the in vivo localization of spiperone analogs (Moerlein et al. 1985). A series of N-alkylated and para-brominated analogs were synthesized so that optimal structural analogs of spiperone could be determined and also so that the effect of the general lipophilicity could help in the design of novel radiopharmaceuticals for the butyrophenone neuroieptics. They found that a high lipophilicity does not completely eliminate the specific binding to the receptors and therefore developing other radiobrominated pharmaceuticals for receptor systems should be possible, GBR halogenated analogues were synthesized as new dopamine uptake carriers (Foulon et al. 1992). The new brominated compound was non-radioactive and the iodinated analogue used [I25I]. The iodinated compound showed good potential for the in vivo investigation of the dopamine uptake carrier and thus the radiobrominated analogue should also be a potential compound for these investigations. A series of studies with [76Br]bromospiperone and [76Br]bromolisuride was conducted by Delforge et al. (1991), Hantraye et al. (1992), Martinet et al. (1990) and Martinet et al. (1991). The two studies by Martinet et al. were conducted on schizophrenic patients by looking at the ratio of radioactivity in the striatum to that in the cerebellum as an index of striatal D2 receptor density. They found no significant difference in the index between schizophrenic patients and normal controls for both compounds. From this they suggest that there is no quantitative abnormality of the D2 receptor density in schizophrenia. The Delforge study investigated a kinetic analysis of bromolisuride binding to the D2 receptor in baboons. The Hantraye study investigated a model of Huntington's disease in the baboon with bromolisuride. 2p-carbomethoxy-3p-(4-bromophenyl)tropane (ß-CBT) was synthesized with [76Br] to investigate its usefulness in imaging dopamine uptake sites in pathological conditions (Maziere et al. 1995a). The compound was synthesized via an electrophilic or nucleophilic substitution. Loc'h et al. (1996a) investigated the substituted benzamides FLB 457 and FLB 463 which were brominated with [76Br] via an electrophilic substitution. They found that FLB 457 has a high uptake in the thalamic structures in the baboon and thus should be a good radioligand for PET investigations of the extrastriatal D2 receptors in the human brain.

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The D1 receptor was studied in vivo in the monkey brain with 11C- and 76Br- labeled NNC 22-0010 (a dopamine antagonist) (Foged et al. 1996). The brominated compound was prepared by a nucleophilic substitution reaction with an iodine precursor and assisted with Cu+. Their findings showed that the compounds have potential for use in imaging the D1 receptor in the human brain. (E)-N-(3-bromoprop-2-enyl)-2p-carbomethoxy-3p-4'-tolyl-nortropane (PE2Br, a cocaine analogue) was labeled with 76Br (Helfenbein et al. 1999). They investigated the compound's pharmacological properties in the rat and PET imaging characteristics in the monkey. The radiobrominated compound was prepared by an electrophilic substitution. The compound was a potent and selective radioligand for the dopamine transporter.

PEPTIDES AND MACROMOLECULES Radiobrominated mouse epidermal growth factor (BrEGF) was investigated (Scott-Robson et al. 1991; Zhao et al. 1999). The production of this compound was optimized as a function of pH. The reaction was performed in an aqueous buffered solution with chloramine-T as the initiator. The compound was selected for this study because the receptor for EGF is over expressed in some tumors. The early work investigated the biodistribution and stability of [76Br]EGF in normal rats compared to [I25I]EGF. Both the iodinated and brominated compounds bound equally in tests with cultured glioma cells and the distribution of both compounds in vivo was similar. BrEGF was coupled to 13 and 46 kDa dextrans to investigate their biodistribution in rats (Zhao etal. 1999). The effect of the dextranation was to prolong the compound's lifetime in the blood. A larger dextran lead to a longer lifetime. The 46 kDa dextran also lowered the kidney accumulation and increased the spleen uptake. The same group has also described the conjugation labeling of macromolecules with 76Br (Yngve et al. 1999). Nsuccinimidyl bromobenzoate (BrNHS) was labeled with 76Br via an electrophilic substitution reaction in the presence of chloramine-T. After purification BrNHS was conjugated to human serum albumin, chromagranin, and 5'-hexylamino-modified oligodeoxynucleotides (ODN) and its phosphorothioate analogues (S-ODN). In this study they used the antisense oligonucleotide sequence for m-RNA encoding for chromogranin in the rat for labeling with the BrNHS. Wu et al. (2000) investigated the distribution of varying sized oligonucleotides conjugated to BrNHS in ex vivo studies in rats. They found that the body distribution was highly dependent on the length of the oligonucleotides. Lengths greater than 20mer are preferable since they do not accumulate predominantly in the kidney. An attempt was made to conjugate octreotide with three compounds: BrNHS, N-succinimidyl 5-bromo-3pyridine-carboxylate and methyl-4-bromobenzimidate (Yngve et al. 2001). The latter compound did not conjugate to octreotide and was not studied further. The former two conjugated compounds were investigated for their binding properties to meningiomas by frozen section autoradiography. BrNHS was found to have poorer binding to meningiomas than the pyridinecarboxylate compound.

MONOCLONAL ANTIBODIES (MAbs) N-Succinimidy para-[77Br]bromobenzoate was synthesized by Wilbur and Hylarides (1991) in order to perform conjugation labeling of an anti-melanoma antibody. A 22% overall yield was obtained. The group demonstrated through an in vitro analysis that the radiobrominated antibody was immunocompetent and retained 80% of the unlabeled immunoreactivity.

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The anti-CEA MAb 38S1, an anti-carcinoembryonic antigen antibody, was labeled with 76Br (76Br-38Sl) with bromoperoxidase in a direct labeling method (Lovqvist et al. 1995). They looked at its biological activity in vitro and compared it to 125I-38S1. They believe their method is a reliable procedure for the production of labeled MAbs and they conclude that the method produces a MAb that has a comparable immunoreactivity to I25I-38SI. This MAb was studied further in a nude rat model with a human colon carcinoma xenograft (Lovqvist et al. 1997a). In the study they compared 76Br-38Sl to 125I-38S1 and found that with 76Br-38Sl after 46 hr postinjection the tumor could still be visualized with PET. However, they did find that the MAb dehalogenated leading to their belief that this causes a higher concentration of free 76Br in the tumor, blood, and other normal tissues than with !25I. Aside from the dehalogenation, they find this compound promising for tumor imaging with PET, In another study, they compared the same MAb to FDG and L-[methyl-11CJmethionine in the same rat model (Lovqvist et al. 1997b). They found that 76Br-38Sl is superior for imaging subcutaneous tumors in the rat model. When they looked at tumor metastases they found no effective difference between 76Br-38Sl and FDG. With promising results for tumor imaging they followed this study up with measuring whole body kinetics of the radiolabeled MAb in pigs with PET (Lovqvist et al. 1999). The data allowed for estimates of the absorbed dose in human organs when calculated with MIRDOSE 3.0 software. The radiation dose was evenly distributed with the highest organ doses being only about two to three times that of the mean absorbed dose. The lungs and liver had the highest doses with about ImGy/MBq. The A33, a humanized IgGl antibody that is another MAb, has also been labeled (Sundin et al. 1999). In previous studies it was found that labeling at physiological pH using chloramine-T was inefficient. They showed a convenient method for labeling with chloramine-T despite these earlier results. The labeling was performed with direct labeling of the antibody giving a 77% labeling yield. The advantage over indirect methods is that the direct method has a higher labeling yield and the advantage over enzymatic labeling is that there is not the difficulty of purifying the antibody from the enzyme. In vitro binding assays were performed with SW1222 colonic cancer cells and the results showed that the immunoreactivity was retained. An indirect method of labeling 76Br-38Sl was investigated by Hoglund et al. (2000). They used N-succinimidyl para-(tri-methylstannyl)benzoate (SPMB) with chloramine-T and then conjugated this compound to the antibody. The total yield was 49%. Immunoreactivity was retained after labeling. This method was investigated since a downside to the direct labeling is that bromide catabolites circulate in the blood giving a high background activity. Also direct bromination occurs mostly on the tyrosine residues and this causes a reduction in the immunoreactivity of the labeled compound. Indirect labeling overcomes this because labeling occurs on the lysine residues.

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Koziorowski J and Weinreich R (1997). Simple preparation of 76Br-, 123I-, and 211At-labeled 5-halo-2deoxyuridine. J. Radioanal. Nucl. Chem. 219, 127–128. Kriss JP, Marayama Y, Tung LA, Bond SB and Revesz L (1963). Fate of 5-bromodeoxyuridine, 5bromodeoxycytidine, and 5-iododeoxycytidine in man. Cancer Res. 23, 260-268. Krohn KA and Knight LC (1977). Radiopharmaceuticals for thrombus detection: selection, preparation, and critical evaluation. Semin Nucl Med 7, 219–228. Kulmala HK, Huang CC, Dinerstein RJ and Friedman AM (1981). Specific in vivo binding of 77Br-pbromospiroperidol in rat brain: a potential tool for gamma ray imaging. Life Sci 28, 1911–1916. Loch C, Halldin C, Bottlaender M, Swahn CG, Moresco RM, Maziere M, Farde L and Maziere B (1996a). Preparation of [76Br]FLB 457 and [76Br]FLB 463 for examination of striatal and extrastriatal dopamine D-2 receptors with PET. Nucl Med Biol 23, 813-819. Loch C, Kassiou M, Strijckmans V, Bottlaender M, Katsifis A, Schmid L, Maziere M, Lambrecht RM and Maziere B (1996b). Pharmacological characterization and positron emission tomography evaluation of 4[76Br]bromodexetimide and 4-[76Br]bromolevetimide for investigations of central muscarinic cholinergic receptors. Nucl Med Biol 23, 235–243. Loch C, Mardon K, Valette H, Brutesco C, Merlet P, Syrota A and Maziere B (1994). Preparation and pharmacological characterization of [76Br]-meta-bromobenzylguanidine (f76Br]MBBG). Nucl Med Biol 21, 49-55. Lovqvist A, Lundqvist H, Lubberink,M, Tolmachev V, Carlsson J and Sundin A (1999). Kinetics of 76Br-labeled anti-CEA antibodies in pigs; aspects of dosimetry and PET imaging properties. Med Phys 26, 249-258. Lovqvist A, Sundin A, Ahlstrom H, Carlsson J and Lundqvist H (1995). 76Br-labeled monoclonal anti-CEA antibodies for radioimmuno positron emission tomography. Nucl MedBiol 22, 125–131. Lovqvist A, Sundin A, Ahlstrom H, Carlsson J and Lundqvist H (1997a). Pharmacokinetics and experimental PET imaging of a bromine-76-labeled monoclonal anti-CEA antibody. J Nucl Med 38, 395–401. Lovqvist A, Sundin A, Roberto A, Ahlstrom H, Carlsson J and Lundqvist H.(l 997b). Comparative PET imaging of experimental tumors with bromine-76-labeled antibodies, fluorine-18-fluorodeoxyglucose and carbon11-methionine. J Nucl Med 38, 1029-1035. Lu L, Bergstrom M, Fasth KJ and Langstrom B (2000). Synthesis of [76Br]bromofluorodeoxyuridine and its validation with regard to uptake, DNA incorporation, and excretion modulation in rats. J Nucl Med 41, 1746-1752. Lu L, Bergstrom M, Fasth KJ, Wu F, Eriksson B and Langstrom B (1999). Elimination of nonspecific radioactivity from [76Brjbromide in PET study with [76Br]bromodeoxyuridine. Nucl Med Biol 26,795802. Lundkvist C, Loch C, Halldin C, Bottlaender M, Ottaviani M, Coulon C, Fuseau C, Mathis C, Farde L and Maziere B (1999). Characterization of bromine-76-labelled 5-bromo-6-nitroquipazine for PET studies of the serotonin transporter. Nucl Med Biol 26, 501–507. Machulla HJ, Marsmann M, Dutschka K and Van Beuningen D (1980). Radiopharmaceuticals. II. Radiobromination of phenylpentadecanoic acid and biodistribution in mice. Radiochem. Radioanal. Lett. 42, 243-250. Machulla HJ, Stocklin G, Kupfernagel C, Freundlieb C, Hock A, Vyska K and Feinendegen LE (1978). Comparative evaluation of fatty acids labeled with C-11, Cl-34m, Br-77, and 1-123 for metabolic studies

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of the myocardium: concise communication. J Nucl Med 19, 298-302. Markey SP, Colburn RW and Kopin IJ (1976). Synthesis and purification of 2-bromo-.alpha.-ergocryptine-82Br. J. Labelled Compd. Radiopharm. 12, 627–630. Mars U, Tolmachev V and Sundin A (2000). Positron emission tomography of experimental melanoma with [76Br]5-bromo-2-thiouracil. Nuclear Medicine and Biology 27, 845-849. Martinet J, Peron-Magnan P, Huret J, Mazoyer B, Baron J, Boulenger J, Loch C, Maziere B, Caillard V and Loo H (1990). Striatal D2 dopaminergic receptors assessed with positron emission tomography and [76Br]bromospiperone in untreated schizophrenic patients. Am J Psychiatry 147, 44–50. Martinot JL, Paillere-Martinot ML, Loch C, Hardy P, Poirier MF, Mazoyer B, Beaufils B, Maziere B, Allilaire JF and Syrota A (1991). The estimated density of D2 striatal receptors in schizophrenia. A study with positron emission tomography and 76Br-bromolisuride. Br J Psychiatry 158, 346-350. Maziere B and Loch C (1986). Radiopharmaceuticals labelled with bromine isotopes. Appl. Radiat. Isot. 37, 703– 713. Maziere B, Loch C, Hantraye P, Guillon R, Duquesnoy N, Soussaline F, Naquet R, Comar D and Maziere M (1984). 76Br-bromospiroperidol: a new tool for quantitative in-vivo imaging of neuroleptic receptors. Life Sc/35, 1349-1356. Maziere B, Loch C, Muller L and Halldin C (1995a). 76Br-beta-CBT, a PET tracer for investigating dopamine neuronal uptake. Nucl Med Biol 22, 993-997. Maziere B, Valette H and Loch C (1995b). 76Br-MBBG, a PET radiotracer to investigate the norepinephrine neurological and vesicular transproters in the heart. Nucl Med Biol 22, 1049–1052. McElvany K, Carlson K, Welch M, Senderoff S and Katzenellenbogen J (1982a). In vivo comparison of 16 alpha[77Br]bromoestradiol-17 beta and 16 alpha-[125I]iodoestradiol-17 beta. J Nucl Med 23, 420–424. McElvany KD, Barnes JW and Welch MJ (1980). Characterization of bromine-77-labeled proteins prepared using myeloperoxidase. Int. J. Appl. Radiat. Isot. 31, 679-688. McElvany KD, Carlson KE, Katzenellenbogen JA and Welch MJ (1983). Factors affecting the target site uptake selectivity of estrogen radiopharmaceuticals: serum binding and endogenous estrogens. J Steroid Biochem 18, 635–641. McElvany KD, Katzenellenbogen JA, Shafer KE, Siegel BA, Senderoff SG and Welch MJ (1982b). 16 alpha[77Br]bromoestradiol: dosimetry and preliminary clinical studies. J Nucl Med 23, 425-430. McElvany KD and Welch MJ (1980). Characterization of bromine-77-labeled proteins prepared using bromoperoxidase. J Nucl Med 21, 953-960. Mercer JR, Xu LH, Knaus EE and Wiebe LI (1989). Synthesis and tumor uptake of 5-82Br- and 5-1311-Iabeled 5-halo-l-(2-fluoro-2-deoxy-beta-D-ribofuranosyl)uracils. J Med Chem 32, 1289–1294. Miholic J, Reilmann L, Meyer H J, Korber H, Dieckelmann A and Pichlmayr R (1992). Estimation of extracellular space and blood volume using bioelectrical impedance measurements. Clin Investig 70, 600–

605. Misra HK, Knaus EE, Wiebe LI and Tyrrell DL (1986). Synthesis of iodine-131, -125, -123 and bromine-82 labelled 5-halo-l-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)uracils. Appl. Radiat. Isot. 37, 901-905. Moerlein SM, Hwang DR and Welch MJ (1988). No-carrier-added radiobromination via cuprous chlorideassisted nucleophilic aromatic bromodeiodination. Int J Rad Appl Instrum [ A] 39, 369-372. Moerlein SM, Laufer P and Stocklin G (1985). Effect of lipophilicity on the in vivo localization of radiolabelled

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spiperone analogues. Int'l J. Nucl. Med. Biol. 12, 353-356. Moerlein SM, Laufer P, Stocklin G, Pawlik G, Wienhard K and Heiss WD (1986). Evaluation of 75Br-labelled butyrophenone neuroleptics for imaging cerebral dopaminergic receptor areas using positron emission tomography. Eur J Nucl Med 12, 211–216. Moerlein SM and Stocklin G (1984). Specific in vivo binding of 77Br-brombenperidol in rat brain. Life Sci 35, 1357–1363. Morkeberg JC, Sheng HP and Huggins RA (1992). Extracellular volume estimation from ratios of bromide to chloride in urine or saliva. Proc Soc Exp Biol Med 199, 68-74. Norton EF, Kondo K, Karlstrom K, Lambrecht RM, Wolf AP and Treves S (1978). Cyclotron isotopes and radiopharmaceuticals. XXVI. A carrier-free separation of bromine-77 from selenium. J. Radioanal. Chem. 44,207-213. Nunn AD and Waters SL (1975). Target materials for the cyclotron production of carrier-free bromine-77. Int. J. Appl. Radiat. hot. 26,731–735. O'Donoghue J A and Wheldon TE( 1996). Targeted radiotherapy using Auger electron emitters. Phys Med Biol 41, 1973-1992. Paans AM, Welleweerd J, Vaalburg W, Reiffers S and Woldring MG (1980). Excitation functions for the production of bromine-75: a potential nuclide for the labelling of radiopharmaceuticals. Int J Appl Radiat hot 31, 267-273. Pierce PA and Peroutka S J (1989). Hallucinogenic drug interactions with neurotransmitter receptor binding sites in human cortex. Psychopharmacology (Berl) 97, 118–122. Qaim SM (1986). Recent developments in the production fluorine-18, bromine-75, -76, -77 and iodine-123. Appl. Radiat. hot. 37, 803-810. Qaim SM, Stoecklin G and Weinreich R (1977). Excitation functions for the formation of neutron deficient isotopes of bromine and krypton via high-energy deuteron induced reactions on bromine: production of bromine-77, bromine-76, and krypton-79. Int. J. Appl. Radiat. hot. 28, 947-953. Qaim SM and Weinreich R (1981). Production of 75Br via the 75Kr precursor: excitation function for the deuteron induced nuclear reaction on bromine. Int J Appl Radiat hot 32, 823-827. Raffel D, Loch C, Mardon K, Maziere B and Syrota A (1998). Kinetics of the norepinephrine analog [76Br]meta-bromobenzylguanidine in isolated working rat heart. Nucl Med Biol 25, 1–16. Rauws AG (1975). Bromide pharmocokinetics: a model for residue accumulation in animals. Toxicology 4,195– 202. Rauws AG and Van Logten MJ (1975). The influence of dietary chloride on bromide excretion in the rat. Toxicology 3, 29-32. Ribeiro MJ, Almeida P, Strul D, Ferreira N, Loch C, Brulon V, Trebossen R, Maziere B and Bendriem B (1999). Comparison of fluorine-18 and bromine-76 imaging in positron emission tomography. Eur J Nucl Med 26, 758-766. Ryser JE, Blauenstein P, Remy N, Weinreich R, Hasler PH, Novak-Hofer I and Schubiger PA (1999). [76Br]Bromodeoxyuridine, a potential tracer for the measurement of cell proliferation by positron emission tomography, in vitro and in vivo studies in mice. Nucl Med Biol 26, 673-679. Sargent T, Kalbhen DA, Shulgin AT, Braun G, Stauffer H and Kusubov N (1975). In vivo human pharmacodynamics of the psychodysleptic 4-BR-2,5- dimethoxyphenylisopropylamine labelled with BRor

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BR. Neuropharmacology 14, 165-174. Scott-Robson S, Capala J, Carlsson J, Malmborg P and Lundqvist H (1991). Distribution and stability in the rat of a 76Br/125I-labelled polypeptide, epidermal growth factor. Int J Rad Appl Instrum B 18, 241–246. Seevers RH, Mease RC, Friedman AM and Desombre ER (1986). The synthesis of non-steroidal estrogen receptor binding compounds labeled with bromine-80m. Nucl. Med. Biol. 13, 483-495. Sihver W, Fasth KJ, Horti AG, Koren AO, Bergstrom M, Lu L, Hagberg G, Lundqvist H, Dannals RF, London ED, Nordberg A and Langstrom B (1999). Synthesis and characterization of binding of 5-[76Br]bromo-3[[2(S)-azetidinyl]methoxy]pyridine, a novel nicotinic acetylcholine receptor ligand, in rat brain. J NeurochemlS, 1264–1272. Soremark R and Ullberg S (1960). Distribution of Bromide in Mice. An Autoradiographic Study with Br-82. Int J Appl Radial Isot 8, 192–197. Strijckmans V, Bottlaender M, Luo H, Ottaviani M, McPherson DW, Loch C, Fuseau C, Knapp FF and Maziere B (1997). Positron emission tomographic investigations of central muscarinic cholinergic receptors with three isomers of [76Br]BrQNP. Eur J Nucl Med 24, 475-482. Suehiro M, Iwamoto M, Arai I and Nozaki T (1990). Bromination, no-carrier-added radiobromination and simultaneously-occurring chlorination by chloramine T. Int J Rad Appl Instrum [ A] 41, 439-447. Sundin J, Tolmachev V, Koziorowski J, Carlsson J, Lundqvist H, Welt S, Larson S and Sundin A (1999). High yield direct 76Br-bromination of monoclonal antibodies using chloramine-T. Nucl Med Biol 26, 923–929. Tolmachev V, Lovqvist A, Einarsson L, Schultz J and Lundqvist H (1998). Production of 76Br by a low-energy cyclotron. Appl. Radiat, hot. 49, 1537–1540. Trepanier LA and Babish JG (1995). Pharmacokinetic properties of bromide in dogs after the intravenous and oral administration of single doses. Res Vet Sci 58, 248–251. USP/NF 25 (2002). The United States Pharmacopeial Convention, Inc., Rockville, MD, 1923–1926. Vaalburg W, Paans AMI, Terpstra JW, Wiegman T, Dekens K, Rijskamp A and Woldring MG (1985). Fast recovery by dry distillation of bromine-75 induced in reusable metal selenide targets via the 76Se(p,2n)75Br reaction. Int. J. Appl. Radiat, hot. 36, 961–964. Vaiseman N, Koren G and Pencharz P (1986). Pharmacokinetics of oral and intravenous bromide in normal volunteers. J Toxicol Clin Toxicol 24, 403–413. Wang SS, Mathis CA and Peroutka SJ (1988). R(-)-2,5-dimethoxy-4-77 bromoamphetamine [77Br-R(-)DOB]: a novel radioligand which labels a 5-HT binding site subtype, [erratum appears in Psychopharmacology (Berl) 1988;96(3):430]. Psychopharmacology (Berl) 94, 431–432. Weinreich R, Alfassi ZB, Blessing G and Stoecklin G (1980). Short-lived neutron deficient bromine isotopes for applications in nuclear medicine. Nuklearmedizin, Suppl. (Stuttgart) 17, 202-205. Wilbur DS and Hylarides MD (1991). Radiolabeling of a monoclonal antibody with N-succinimidyl para[77Br]bromobenzoate. Int J Rad Appl Instrum B 18, 363-365. Wong S and Ache HJ (1976). On the preparation of 80Br- or 82Br-biomolecules via excitation labelling methods. Int J Appl Radial hot 27, 19–25. Wu F, Yngve U, Hedberg E, Honda M, Lu L, Eriksson B, Watanabe Y, Bergstrom M and Langstrom B (2000). Distribution of (76)Br-labeIed antisense oligonucleotides of different length determined ex vivo in rats. EurJ Pharm Sci 10, 179–186. Xing T, Wu F, Brodin O, Fasth K J, Langstrom B and Bergstrom M. (2000). In vitro PET evaluations in lung

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cancer cell lines. Anticancer Res 20, 1375–1380. Yasui LS, Hughes A and DeSombre ER (2001). Relative biological effectiveness of accumulated 125IdU and 1251-estrogen decays in estrogen receptor-expressing MCF-7 human breast cancer cells. Radiat. Res. 155, 328-334. Yngve U, Hedberg E, Lovqvist A, Tolmachev V and Langstrom B (1999). Synthesis of N-succinimidyl 4[76Br]bromobenzoate and its use in conjugation labelling of macromolecules. Acta Chem. Scand. 53, 508– 512. Yngve U, Khan TS, Bergstrom M and Langstrom B (2001). Labelling of octreotide using 76Br-prosthetic groups. J. Labelled Compd. Radiopharm. 44, 561–573. Zhao Q, Tolmachev V, Carlsson J, Lundqvist H, Sundin J, Janson JC and Sundin A (1999). Effects of dextranation on the pharmacokinetics of short peptides. A PET study on mEGF. Bioconjug Chem 10,938– 946.

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15. DEVELOPMENT OF RADIOLABELED PROBES TO MONITOR GENE THERAPY CHYNG-YANN SHIUEA AND STEPHEN L.ECKB Department of Radiology, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USAA; Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA.8

INTRODUCTION Not long after the initial characterization of DNA, scientists speculated that alterations in human gene expression could be used for therapeutic benefit (Tatum, 1966). In the last decade, this has materialized in a myriad of gene therapy clinical trials for a variety of inherited and acquired disorders (Eck & Wilson, 1996). These are all based on a common strategy to use gene delivery to effect local production of a therapeutic protein in a target tissue. This general approach circumvents the inherent problem of delivering complex proteins to highly special tissues and subcellular compartments and exploits the tremendous stability and plasticity of the introduced gene sequences. "Gene Therapy: it's real, it works and it's coming to your practice" was displayed on the cover of a Medical Grand Rounds Press publication in 1993 well before this technology had been reduced to a standard therapeutic modality: a still somewhat elusive goal. The principal roadblock stems from gene therapy's reliance on a targeted drug delivery, wherein the drug is the gene of interest and a vector is used to package the genetic material to facilitate its delivery, uptake and expression in the desired target tissue. In general targeted drug deliveries have been difficult to implement as evidenced by the few commercially available agents that are specifically targeted to an organ or tissue. Unlike conventional drug development that is well supported by highly developed pharmacologic tools and principles, gene therapy has lacked this level of developmental support until recently, when new imaging technologies have begun to emerge. These technologies will serve to answer questions about the distribution, extent and duration of gene expression that have been inadequately addressed up to now and are pivotal to the future development of this field. The difficulty in assessing the efficiency of gene transfer in patients and thereby appreciating potential limitations of genetic vector designs can be appreciated by examining the currently employed techniques that are used in current phase I, II and III clinical trials. To date, the only reliable method available has been the biopsy of tissue with subsequent analysis for the therapeutic transgene DNA, RNA, and/or protein. This approach has several shortcomings: (1) it is associated with some degree of risk and discomfort in acquiring the tissue. This is especially relevant to CNS gene therapies: an area of intense interest for malignant and neurodegenerative diseases. Biopsies of the brain or brain tumor are costly and have significant risk. (2) The biopsy can perturb the tissue under study and in the case of CNS studies, alter the extent of enhancement on MRI: a widely used clinical assessment for CNS disease. (3) There are inherent sampling errors since gene expression may be unevenly distributed, making even crude quantification of gene expression difficult. (4)

Handbook of Radiopliarmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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Only a few biopsies (at most) can be done in any one patient thus raising the likelihood that the relevant time points will be missed. (5) The results of biopsy studies provide only limited descriptive data on gene expression and therapeutic protein activity since they rely on histopathologic techniques that lack the dynamic component of pharmacologic assessments. (6) Biopsies will not reveal any information about unintended gene transfer into tissues that were not suspected of harboring the vector and therefore were not biopsied. This is particularly important when considering the possibility of germline gene transfer. For example, determination of whether vector administration results in inadvertent transduction of gonadal tissues is limited to necropsy studies in experimental animals. Confirmation or repudiation of such finding in humans cannot be done with existing approaches since biopsies of testes and ovaries are not attractive options in patients. As an alternative to direct assessment of tissue specific expression, several strategies have been devised to provide surrogate markers that can report the fate of a transferred gene. For example, the therapeutic protein can be expressed in parallel with a secreted marker protein, which is in turn assessed using serial blood samples. Both human a-fetoprotein (O'Neal et al., 2000) and the ß-chain of chorionic gonadotropin (ß-HCG) (Zoltick & Wilson, 2000) have been tested in animal models for this application. The marker gene and therapeutic gene can be tightly linked to insure coordinate expression. Ideally, the marker proteins are biologically inert and readily assayed using convenient serum assays as is the case with a-fetoprotein and pHCG. Nonetheless, they will provide only limited information on the co-expressed therapeutic gene. No information can be gained regarding the tissue distribution of the therapeutic gene since one cannot ascertain whether the secreted marker protein is coming from a small foci of cells or from a widely dispersed group of cells within the target tissue. Moreover, differences in the rates of clearance of the marker protein and the rate of turnover of a cell bound therapeutic protein may obscure important differences in their duration of expression. Quantitative Real-time PCR has been proposed as a method to measure gene distribution in vivo (Hackett et al., 2000). In addition to requiring tissue at multiple time points and the attendant problems noted above, this only addresses the identity and integrity of the inserted gene, and does not reveal whether the gene continues to be expressed. Our own work has aptly illustrated how conventional imaging techniques such as MRI can be misleading with respect to the clinical course of the therapy. A 42 year old woman with a malignant glioma was treated with intratumor injection of an adenovirus expressing herpes virus thymidine kinase (Eck et al., 19%; Alavi et al., 1998). One month later she developed an increasing area of enhancement on gadolinium enhanced MRI which was radiographically indistinguishable from the anticipated re-growth of her tumor. Evaluation using 18 FDG-PET imaging reveal that the area of interest had low uptake of 2-[ F]FDG, in contrast to her pretreatment PET image of the tumor. While this was reassuring that the MRI was not revealing new tumor growth (she remained disease free for three more years), it provided no information as to whether the inserted genetic material was continuing to function. Overall, the absence of technology to assess the distributions and pharmacokinetics of the gene therapy drugs (encoded therapeutic protein) has greatly hindered the clinical developments in this field. As a result, empiric clinical trial designs are set forth and then altered with little tangible data to guide the rational improvement in potentially promising therapies. Therefore, it is critically important to have a non-invasive imaging modality which offers the possibility of monitoring the location, magnitude, and duration of gene expression for in vivo use in humans.

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Currently, several imaging methods are under active investigation as tools for studying gene expression in living subjects. These include optical imaging (Contag et al., 1998)), MRI (Bogdanov & Weissleder, 1998), SPECT (Tjuvajev et al., 1996) and PET (Tjuvajev et al., 1998; Gambhir et al., 1998a, 1999a; Hustinx et al., 2001), Conventional approaches using green fluorescent protein (Misteli & Spector, 1997) and luciferase (Jacobs et al., 1993) to locate the reporter gene expression in animals is inapplicable in humans without resorting to biopsy based methods. Recently, MRI has been used to indicate reporter gene expression in living animals (Louie et al., 2000). This approach uses a contrast agent that is enzymatically processed by ßgalactosidase. When the lac Z reporter gene is expressed, it leads to the expression of p-galactosidase, which hydrolyzes a gadolinium contrast agent. The hydrolysis product provides complexed gadolinium that increases proton relaxation times with subsequent changes in MRI signal. This approach has not yet been implemented in large animals in which many obstacles exist. Moreover, it should be noted that MRI has a -4

-5

sensitivity of ~ 10

to 10 M compared to nuclear imaging techniques (PET and SPECT) which have

sensitivities of ~ 10

to 10 M of radiolabeled substrate. Nuclear imaging techniques offer the highest level

of sensitivity for imaging relatively low levels of reporter gene expression and provide the possibility of monitoring the location, magnitude, and duration of reporter gene expression for in vivo use in living animals and humans. Such noninvasive, repetitive and quantitative imaging techniques will facilitate human gene therapy trials and allow for the study of animal models of human disease, provided the radiolabeled substrate activity can be linked to gene expression and biologic activity. The in vivo imaging of gene expression can be directed either at genes that are transferred into cells of organ systems (transgenes) or at endogenous genes. Imaging transgenes can monitor the expression of genes that are transferred into cells to study the normal regulation of gene expression or to produce a therapeutic outcome. This is conceptually and technically much easier since the imaging modality can be built into the therapeutic gene construct. Imaging endogenous gene expression is more challenging since there are more biologic constraints on the system. Imaging of endogenous gene expression provides the means to examine changes in gene expression during development, aging, and environmental stimulation, as well as those changes that occur when a normal phenotype changes to a disease phenotype or changes in response to therapy. Of the two approaches to imaging gene expression, imaging endogenous expression is potentially more broadly useful. It has the capacity to detect the genetic changes that underlie the normal function of cells, as well as detect the alterations of gene expression that initiate disease (Phelps, 2000). Imaging endogenous gene expression can be directed at either transcription of genes into mRNA or the subsequent production of a protein. In the former, the ribonucleotide coding sequence (mRNA) is the target for imaging and a modified radiolabeled antisense oligodeoxynucleotide (RASON) coded with the complement of the sequence of a single strand of mRNA is the imaging probe. The principles and requirements of this imaging approach were recently reviewed in detail (Gambhir et al., 1999b). A significant limitation of this approach is the biodistribution of the RASON and the specificity of its binding to the target mRNA. Imaging transgene expression typically involves a reporter gene and a reporter probe. The reporter gene is linked to the therapeutic gene by a common promoter to insure the linked transcription and translation of the

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therapeutic and reporter elements. The promoter initiates transcription of the gene. Currently, two reporter gene/reporter probe approaches have been used to image transgene expression. In the first approach, the reporter protein is an enzyme, and the reporter probe is a radiolabeled enzyme substrate. In the second approach, the reporter protein is a receptor, and the reporter probe is a receptor ligand. REPORTER GENE The ideal reporter gene should have the following characteristics: (1) The reporter gene protein should produce specific reporter probe accumulation only in those tissues in which it is expressed; (2) When the reporter gene is not expressed, there should be no significant accumulation of reporter probe in tissues; (3) No significant immune reaction to the reporter gene product; (4) The size of the reporter gene and the promoter driving it are limited by the capacity of the delivery vehicle, and (5) The reporter gene assay should correlate well with levels of endogenous gene expression. REPORTER PROBES Monitoring transgene expression requires the appropriate combination of reporter genes and reporter probes. Reporter genes and vectors have been reviewed elsewhere (Smith, 1995; Verma & Somia, 1997) and will not be discussed in detail here. The focus of this chapter will be on the development of reporter probes. In contrast to radiopharmaceuticals used for studying blood flow, glucose metabolism and receptor binding (Phelps el al., 1986; Diksic & Reba, 1991), the development of probes for monitoring gene expression with PET and SPECT is still in its infancy and has been reviewed in great details (Gambhir et al., 1999b,c; Gambhir et al., 2000). Similar to radiopharmaceuticals used for blood flow, metabolism and receptor binding studies, the ideal reporter probes for monitoring the gene expression would have the following characteristics: (1) Stable in vivo; (2) Cleared rapidly from the blood and non-specific sites in tissues; (3) Conveniently radiolabeled with a variety of radionuclides without significant change in its properties; (4) Non cytotoxic; (5) Able to reach the area(s) of interest without transport across membranes being a limitation, and (6) Image signal should correlate well with levels of gene protein in vivo. Based on these characteristics, two classes of compounds have been used to date as reporter probes for non-invasively monitoring transgene expression in vivo: (1). Enzyme-based substrates, and (2). Receptor-based radioligands. The high specific activity of the probe and the signal amplification are the key issues in gene expression imaging since a low level of gene expression may have significant biological and clinical effects. Higher levels of imaging signal per unit level of gene expression will lead to a higher sensitivity for the assay. The advantage of enzyme-based substrates approach is that one molecule of enzyme can metabolize and trap many reporter probe molecules, leading to signal amplification. The receptor-based approach is primarily a fixed interaction of binding of one or a few ligand molecules to one receptor. The enzymes that have been investigated for gene expression imaging include cytosine deaminase (Haberkorn et al., 1996), ß-galactosidase (Louie et al., 2000), tyrosinase (van Langevelde et al., 1988; Weissleder et al., 1997) and Herpes simplex virus thymidine kinase (HSV1-TK) (Wiebe et al., 1997; Tjuvajev et al., 1995; Haberkorn et al., 1997; Gambhir et al., 1998a; Hustinx et al., 2001). Among these enzymes, HSV1-TK is the most extensively studied.

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The receptors that have been investigated for gene expression imaging to date are dopamine D2 receptor (MacLaren et al., 1999) and the peptide receptors (Rogers et al., 1997; Zinn et al, 2000). 1. SUBSTRATES FOR HSV1-TK For metabolic-trapping, two main categories of substrates have been investigated as reporter probes for imaging HSV1-TK expression: uracil analogs labeled with radioactive iodine (Tjuvajev et al., 1996; Morin et al., 1997a,b; Wiebe et al., 1997) and acycloguanosine analogs labeled with fluorine-18 (Alauddin et al., 1996, 1998; Barrio et al., 1997; Monclus et al., 1997a,b; Shiue et al., 1999; Namavari et al, 2000). These two classes of substrates are phosphorylated by HSV1-TK to their monophosphates and metabolically trapped in cells. The chemical structures of the most commonly used radioactive labeled uracil analogs as gene expression imaging agents are shown in Figure 1.

OH l.R,-I,R 2 -F,R 3 -H, FIAU 2. R i =ICH-CH-, R 2=H, R 3-F, IVFRU

Figure 1. Chemical structures of uracil analogs as gene expression imaging agents. Similar to hexokinase, which phosphorylates 2-deoxyglucose (DG) and 2-deoxy-2-fluoro-D-glucose (FDG) to the corresponding DG and FDG-6-phosphates and traps them intracellularly (Sokoloff et al., 1977; Gallagher et al., 1978), HSV1-TK phosphorylates both uracil and acycloguanosine analogs to their monophosphates leading to intracellular accumulation. Endogenous mammalian TK phosphorylate these agents much less efficiently. Cellular retention of radioactivity is therefore an indicator of HSV1-TK gene expression. Before their use as gene expression imaging agents, several of these agents have been used as viral infection imaging agents. Autoradiographic studies in rats with an anti-viral agent, carbon-14 labeled 5-methyl-2'fluoroarabinouridine (FMAU) demonstrated that FMAU was phosphorylated to its monophosphate and trapped in those tissues expressing HSV1-TK (Saito et al,. 1982, 1984). Later, 2'-fluoro-2'-deoxy-l-p-Darabinofuranosyl-5-iodouracil (FIAU) was also used as a viral infection imaging agent (Misra et al, 1986; Tovell et al, 1988). FIAU was considered to have good imaging potential because of its in vitro HSV1-TK sensitivity and selectivity and its in vivo stability characteristics. Thus, FIAU labeled with carbon-14 (Tjuvajev et al., 1995), iodine-131 (Tjuvajev et al., 1996, 1999), and recently iodine-124 (Tjuvajev et al., 1998) have been investigated as agents for imaging HSV-TK gene expression in animals bearing RG2 tumors

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with autoradiography, gamma camera/single photon emission computed tomography (SPECT), and PET, respectively. These studies demonstrate that tumors with various levels of HSV1-TK gene expression can be visualized by PET imaging 32 hours after injection of [

124

I]FIAU. Highly significant correlations between the level of

124

[ I]FIAU accumulation and HSV1-TK mRNA levels as well as the sensitivity (IC5Q) of corresponding cell lines to ganciclovir were observed. Hence, FIAU is a potential imaging agent for monitoring HSV1-TK gene expression in transduced tumors in animals. There are concerns about the potential toxicity associated with the administration of radiolabeled FIAU for diagnostic studies in patients (Tjuvajev et al., 1996) due to two deaths that were associated with a clinical trial evaluating the long-term administration of FIAU as a potential anti-viral agent in treatment of patients with chronic hepatitis-B virus infection (Mckenzie et al., 1995). 124

However, the calculated mass of a 4 mCi dose of no-carrier-added [ I]FIAU for PET studies in patients is only 47 ng (Gambhir et al., 2000) which represents only 1/75,000 to 1/2,530,000 of the daily dose that was administered to patients in the 14- to 28-day hepatitis B clinical trials, where no FIAU-related toxicity was observed (Mckenzie et al., 1995). Deiodination and positron yield of iodine-124 (24% for iodine-124 v.s. >97% for fluorine-18, Pentlow et al., 1996) may be factors to consider before using iodine-124 labeled substrates as PET imaging agents. A recent study showed that in the unlesioned cat brain as well as in the 124 normal human brain, the uptake of [ IJFIAU was low (< 0.02%/g and < 0.0002%/g, respectively). However, 124

in a recurrent glioblastoma patient, the uptake of [ I]FIAU in the brain was relatively high (0.001 %/g ,5-10 124 minutes after injection) and cleared slowly over 68 hours (Jacobs et al., 2001). This suggests that [ IJFIAU may be useful for imaging HSV1-TK gene expression within brain tumors with BBB disruption or in organs outside the CNS. Other uracil analogs that have been evaluated as substrates for HSV1-TK in cell culture include 5-(2[125I]Iodovinyl)-2'-deoxyuridine([125I]IVDU), 5-(2-['25I]iodovinyl)-2'-fluoro-2'-deoxyuridine ([!25I]IVFRU), 5-(2-[ I]iodovinyl)-2'-fluoro-2'-deoxyarabinouridine([ I]IVFAU),and 5-(2-[ I]iodovinyl)-arabinouridine ([ I]IVAU)(Morin et al., 1997a) (Figure 2). Structure-activity relationships studies indicate that the uptake of these compounds in KBALB-STK cells is variable and highly dependent on the nature of the sugar 2'substituent. When a fluoro or a hydroxy substituent is present in the arabinofuranosyl configuration at the 2'position, there is diminished cellular uptake in KBALB-STK cells relative to hydrogen or fluorine in the ribofuranosyl configuration. [ IJIVFRU, in particular, has been shown as a promising agent for detecting HSV1-TK expression using scintigraphy (Morin et al., 1997b; Wiebe et al., 1997).

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OH

l . R ! = H , R 2 = H , IVDU 2. R , = H , R 2 = F , IVFRU 3. R,=F, R 2=H, IVFAU 4. Rt=OH, R 2=H, IVAU

r!25

Figure 2. Chemical structures of (E)-5-(2-[

I]iodovinyl)-2'-deoxyuridine and its analogs.

In addition to radiolabeled uracil analogs, other anti-viral agents, such as ganciclovir and its analogs (Alrabiah & Sacks, 1996) have also been evaluated as potential agents for imaging HSVI-TK gene expression. Initially, 8-[ H]ganciclovir was evaluated in cell culture and was found to accumulate in cells expressing HSVI-TK compared to control cells (Gambhir et a/.,1998a; Haberkorn et al., 1997, 1998; Tjuvajev et al., 1995). Further studies showed that 8-[ H]ganciclovir has low affinity for HSVI-TK and its uptake into cells is relatively slow (Haberkorn et al., 1998). Nevertheless, ganciclovir and its analogs were labeled with fluorine-18 and evaluated as potential agents for imaging HSVI-TK reporter gene expression using PET. There are two approaches to label acycloguanosine with fluorine-18: (1) Electrophilic fluorination of guanine 18

moiety with [ F]F2, and (2) Nucleophilic substitution of a good leaving group in the side-chain with IO

1Q

[ Fjfluoride. The first approach was illustrated in the synthesis of 8-[ F]fluoroguanine analogs in low yield (~1 % at end of bombardment, BOB) and low specific activity (2 Ci/mmol at end of synthesis, EOS) (Scheme 1) (Namavari et al., 2000).

HANDBOOK OF RADIOPHARMACEUTICALS

474

o

H,N

l.R-

2. R

3.R-

Ganciclovir

CH HO

Penciclovir

OH

Scheme 1. Synthesis of 8-[ FJfluoroguanine analogs. 18

Among these probes, initial cell culture studies showed that 8-[ FJfluoroacyclovir had low uptake in cells with low levels of HSV1-TK gene expression and was, therefore, not studied further (Srinivasan et at., 1996). 14 18 Cell culture studies with 8-[ C]ganciclovir and 8-[ F]fluoroganciclovir showed significant accumulation in TK expressing cells compared to cells that did not express TK (Gambhir et al., 1998a). Additionally, levels of HSV1 -TK mRNA and levels of HSV1 -TK enzyme correlated well with accumulation of 8-[ C]ganciclovir 18 and 8-[ F]fluoroganciclovir. MicroPET and digital whole body autoradiography studies in mice demonstrated that mice that received adenovirus expressing HSV1-TK showed significant 818 [ F]fluoroganciclovir liver retention consistent with adenovirus-mediated hepatic expression of TK. There 18 was excellent correlation with the 8-[18 F]fluoroganciclovir %ID/g liver for both HSV1-TK mRNA and 18 HSV1-TK enzyme. In addition, 8-[ FJfluoroganciclovir is stable in vivo as > 98% of the activity excreted in mouse urine is the parent compound at 60 minutes postinjection. However, in comparison, 818 14 [ F]fluoroganciclovir has a lower sensitivity for imaging HSV1 -TK gene expression than 8-[ CJganciclovir. 18 The binding affinity of 8-[18 F]fluoroganciclovir for HSV1-TK is about 10 times lower than that of 8[14 C]ganciclovir. Penciclovir, like ganciclovir, is also phosphorylated selectively by HSV1-TK to its monophosphate and trapped intracellularly (Boyd et al., 1993). Initial cell culture studies indicate that the uptake of 8[ HJpenciclovir in cells is higher compared to 8-[ HJganciclovir (Gambhir et al., 1998b). To improve the 18 sensitivity of the HSV1 -TK reporter gene, 8-[ FJfluoropenciclovir was synthesized and showed to have two18 to three-fold advantage over 8-[18 F]fluoroganciclovir (Iyer et al., 2001). However, similar to that of 8Ig 1Q [ FJfluoroganciclovir, 8-[ FJfluoropenciclovir has a lower binding affinity for HSV1-TK compared to penciclovir itself.

475

DEVELOPMENT OF RADIOLABELED PROBES TO MONITOR GENE THERAPY An alternate approach is to use nucleophilic substitution of a good leaving group in the side-chain of jo

ganciclovir and penciclovir to introduce the F label. This approach has been used to prepare two potent 18

HSV1 -TK gene expression imaging agents, namely, 9-[(3-[ F]fluoro-l-hydroxy-2-propoxy)methyl]guanine 18

or FHPG, and 9-[4-f F]fluoro-3-(hydroxymethyl)butyl]guanine or FHBG in high yield with high specific activity (Alauddin et al, 1996, 1998, 1999; Monclus et al., 1997a; Shiue et al., 1999) (Scheme 2).

18 1).K[F]/K2.2.2

HN

2). H + orOH

MTrHN

OTs

MT

1. X=O

FHPG

2. X=CH2 FHBG Scheme 2. Synthesis of FHPG and FHBG. These approaches produce FHPG or FHBG as a racemic mixtures. However, pure (R)- and (S)- 9-[(3jo

f F]fluoro-l-hydroxy-2-propoxy)methyl]guanine were synthesized (Monclus et al., 1997b), evaluated in 18 vitro and found to be no different in their pharmacological activity. Accumulation of (R)-[ FjFHPG, (S)10

10

[ FjFHPG and rac-[ F]FHPG on 9L cells expressing HSV1 -TK are also comparable to each other (Monclus 18

et al., 1999a). In vivo studies in rodent showed that the uptake of rac-[ F]FHPG in tumors transduced with HSV1-TK is higher than that of nontransduced tumors (Alauddin et al., 1999; Monclus et al., 1999b, Hustinx et al., 2001) and that tumors transduced with HSV1-TK can be imaged with PET (Hustinx et al., 2001). 18

Racemic [ F]FHBG was also synthesized and shown to have higher uptake in HT-29 cells compared to [I8F]FHPG; the uptake ratio of [18F]FHBG to [18F]FHPG for transduced HT-29 cells and nontransduced HT29 cells is 18.2 and 8.5, respectively (Alauddin et al., 1998). Pharmacokinetic studies in monkeys showed 1R

18

18

1 ft

that rac-[18F]FHPG, and probably (R)-[18 F]FHPG, (S)-[ F]FHPG and rac-[ F]FHBG, have a favorable radiation dosimetry for sensitive organs. They are stable in vivo, lack recirculating labeled metabolites and are predominantly excreted through the kidneys (Bading et al., 1997; Monclus et al.,1999b). Additionally, FHPG and FHBG do not have decreased affinity for HSV1-TK compared to the parent compounds and do not have any significant cell cytotoxicity at the concentrations used (Gambhir et al., 2000). Therefore, fluorine-18 labeled FHPG and FHBG may serve as useful probes for imaging gene expression in humans. We have developed a simplified method to prepare both [18F]FHPG and [18F]FHBG in 15-20% yield in a synthesis time of 60-70 minutes from end of bombardment (Shiue et al., 1999, 2000). The advantages of this simplified method are that it eliminates HPLC purification, has higher yield than the HPLC purification

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476

method, and the method is readily amenable to automation. The disadvantage of this method, however, is that the final product contains a small amount (5-30 jig) of non-radiolabeled ganciclovir or penciclovir as byis product. In spite of this, [ F]FHPG synthesized by this simplified method has a similar uptake in glioma 9L cells as that prepared by HPLC purification (Shiue et al., 1999). It is feasible for PET imaging of HSV1-TK gene transfer to tumors and is highly specific for HSV1-TK expression (Hustinx et al., 2001). 18

18

[ FJFHBG is more lipophilic than [ FJFHPG which may partially account for the differences in their uptake in HT-29 cells ( Alauddin et al., 1998). The partition coefficients of [I8F]FHBG and ['8F]FHPG are 0.165±0.023 and 0.126±0.022, respectively. [18F]FHBG is more stable than [18F]FHPG in acidic medium due 18

to the hemiaminal linkage in the side chain of [ F]FHPG that facilitates the cleavage of the acyclic C-N bond 18 to yield 3-[ F]fluoro-l ,2-propanediol as a radioactive by-product (Scheme 3) (Shiue et al., 1999). O

HoN

Scheme 3. Decomposition of [ F]FHPG in acidic medium. COMPARISON OF URACIL AND ACYCLOGUANOSINE ANALOGS AS GENE EXPRESSION IMAGING AGENTS Both uracil and acycloguanosine analogs were developed originally as anti-viral agents (Alrabiah & Sacks, 1996) and were used later as reporter probes for imaging gene expression. Several reports have demonstrated the utilities of fluorine-18 labeled 8-FGCV, 8-FPCV, FHPG, FHBG and iodine-123/125/131/124 labeled FIAU and IVFRU as reporter probes for imaging gene expression (Alauddin et al., 1996, 1998, 1999; Blasberg & Tjuvajev, 1999; Monclus et al., 1997a; Namavari et al., 2000; Shiue et al., 1999; Wiebe et al., 1997). Unfortunately, these reports all used different cell lines and various levels of HSV1-TK gene expression. Therefore, it is difficult to compare the advantages and disadvantages of these reporter probes. A direct detailed comparison of all these radiolabeled probes for imaging HSV1-TK reporter gene expression is highly desirable. In vitro studies showed that the uptake of FHBG in HT-29 cells was two-fold higher than that of FHPG ( Alauddin & Conti, 1998). In vitro and in vivo studies also showed a two- to three-fold advantage of 8-FPCV over 8-FGCV (Iyer et al, 2001). Direct comparison between lUdR, GCV and FIAU showed that FIAU is a substantially better marker substrate for the HS V1 -TK enzyme than IUdR or GCV

DEVELOPMENT OF RADIOLABELED PROBES TO MONITOR GENE THERAPY ( Tjuvajev et al, 1995). Recently, a direct comparison of the uptake of 8-FGCV, 8-FPCV, FHPG, FHBG and [14C]FIAU in C6 cells expressing HSV1-TK showed that FIAU and FHBG are the better candidates for imaging HSV1-TK reporter gene expression (Gambhir et al., 2000). However, the true utility of these HSV1 TK probes must ultimately be evaluated in vivo. Future studies that directly compare uracil and acycloguanosine analogs in vivo, using the same tumor models and under equal levels of HSV1-TK expression, will help to better define the advantages and disadvantages for each probe. 2. RADIOLIGANDS FOR RECEPTOR BINDING There are several radioligands available for imaging various receptor systems including dopamine, benzodiazepine, serotonin and cholinergic receptors among others. However, only two receptor systems, namely dopamine D2 and somatostatin receptors have been investigated as reporter gene for in vivo reporter gene imaging. Two classes of compounds with high affinity to dopamine D2 receptor have been investigated as dopamine D2 receptor ligands: butyrophenones (Wagner et al., 1983; Shiue et al., 1985, 1986, 1987; Moerlein et al., 1985; Coenen et al., 1987; Chi et al., 1986; Kiesewetter et al., 1986; Welch et al, 1986; Satyamurthy et al, 1986) and benzamides (Ehrin et al., 1985; Kessler et al, 1991) (Figure 3).

HQ

CONH-Crif J^ PCH3

Raclopride (Benzamides)

N-Alkylspiperone (Butyrophenones) Figure 3. Chemical structures of dopamine D2 receptor ligands.

478

HANDBOOK OF RADIOPHARMACEUTICALS 18

Among these radioligands, 3-(2'-[ F]fluoroethyl)spiperone was used as a reporter probe and dopamine E>2 as a reporter gene to image reporter gene expression following somatic gene transfer (MacLaren et al., 1999). The other receptor used as a reporter gene for imaging reporter gene expression is somatostatin receptor. Using this reporter gene (hSSTr2) and a ""^Tc-labeled somatostatin analog (P2045) as a reporter probe, it was feasible to image expression of the hSSTr2 reporter gene (Zinn et al.,. 2000). Since these reporter genes express endogenous human proteins, their expression should not invoke an immune response. By comparison, we have observed antibody responses to HSV1-TK following adenoviral delivery of HSV1-TK in our own clinical studies. 3. ANTISENSE OLIGODEOXYNUCLEOTIDES Antisense oligonucleotides are attractive imaging agents due to their potential specificity and the theoretic ability to target any gene. However, they are the most challenging and the least explored (Hnatowich, 1999). Labeled antisense probes could be used to bind to and illuminate specific mRNAs and thereby report gene expression. Messenger RNA concentrations are typically in the range of 1 to 1000 pmol/L (Hargrove et al., 1990). The binding affinity of antisense drugs for mRNA is very high. However, a single mismatch can drop the binding affinity by as much as 300-fold (Crooke & Lebleu, 1993). It is estimated that mRNA concentrations as low as 1 pmol/L tissue can probably be imaged with PET using radiolabeled antisense oligodeoxynucleotide (RASON) probes with specific activity of 1-10 Ci/nmol/L (Pan et al., 1998). Oligodeoxynucleotides are more stable in vivo compared to oligoribonucleotides (Wickstrom, 1986) and therefore are the targets for drug and imaging probe development. Biodistribution of Oligodeoxynucleotides in normal animals has been reported (Crooke, 1995). Whole-body autoradiography has also been used to study the pharmacokinetics, metabolism and elimination of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A) in mice (Phillips et al., 1997). The first RASON probe to be developed for imaging endogenous gene expression was an indium-labeled 15mer oligodeoxynucleotide targeted against the amplified c-myc oncogene (Dewanjee et al., 1994). This study demonstrated rapid accumulation of the imaging probe in a tumor expressing the c-myc oncogene (2 hours after injection) compared with a control tumor. Oligodeoxynucleotides labeled with technetium-99m (Hnatowich et al., 1995) and iodine-125 (Cammilleri et al., 1996) have also been reported. Most recently a general method for the synthesis of Oligodeoxynucleotides (phosphodiester, phosphorothioate and 2'-Omethyl oligodeoxynucleotide) labeled at 3' end with fluorine-18 and their biodistributions in baboons were reported (Dolle et al., 1997; Tavitian et al., 1998) (Figure 4). This study showed that (1) each of the three types of fluorine-18-labeled Oligodeoxynucleotides studied behaved very differently in vivo, (2) the 3' labeling with fluorine-18 did not affect the biodistribution of each probe; and (3) PET could be used to quantitate the biodistribution of Oligodeoxynucleotides in vivo. However, this study did not attempt to target any particular mRNA for imaging gene expression. Another fluorine-18 labeled antisense oligodeoxynucleotide, 5'-deoxy-5'-['8F]fluoro-oligodeoxynucleotide (Figure 4) was also synthesized and showed that the replacement of 5'-OH by a fluorine atom did not cause any significant changes in hybridization affinity to complementary RNA sequence (Pan et al, 1998, 1999). Further validation and continued exploration are needed to use antisense approach for imaging gene expression.

O S

IP

O

Oligodeoxynucleotide

O~

O—P-O—C G C C A G C T I! O Figure 4. Chemical structures of fluorine-18 labeled oligodeoxynucleotides. CONCLUSION Assessment of gene expression by nuclear medicine techniques is in an early stage of development and has yet to be established in patients. Assessing gene expression following gene therapy has the potential to greatly advance gene therapy applications, which currently are limited by gene delivery technologies. As better genetic vectors are developed, the parallel development of imaging technology will allow more precise assessments of the distribution and duration of gene expression. Pivotal to the success of imaging therapeutic gene expression will be the ability to reliably correlate the measured effects with clinical benefit. One might easily envision that a very sensitive PET technique might detect levels of gene expression that were not clinically important. The converse could also be true. However, gene therapy offers an ideal setting in which to develop this technology, as there is an inherently high level of contrast compared to other applications of this technology that might be envisioned. For example, the therapeutic expression of HSV1-TK introduces an enzyme that is only present in the transduced tissues since there is normally no endogenous expression of this protein. Moreover, the radiolabeled substrates for HSV1-TK do not need to compete with endogenous substrates to any significant extent for phosphorylation and retention. This stands in contrast to the wide spread endogenous expression of glucohexokinase and its widely available substrate that form the basis of conventional FDG PET scans. Assessing endogenous gene expression posses a more formidable challenge for just these same reasons. However, there are clinical circumstances where sufficiently different levels of gene expression may afford the opportunity to detect clinically meaningful differences in gene expression. The most striking examples will likely be found in infectious diseases and in neoplasms. The progress to date has established the principles needed to implement these technologies. Early clinical trials are likely to follow soon and will provide a clearer picture of the obstacles to successfully introducing these strategies into routine clinical practice.

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HANDBOOK OF RADIOPHARMACEUTICALS

ACKNOWLEDGEMENT This work was supported by funds from NIH grants RO1 CA 85765 and PO1 CA 66726.

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Louie AY, Huber MM, Ahrens ET, Rothbacher U, Moats R, Jacobs RE, Eraser SE and Meade TJ (2000) In vivo visualization of gene expression using magnetic resonance imaging. Nature Biotech., 18, 321325. MacLaren DC, Gambhir SS, Satyamurthy N, Barrio JR, Sharfstein S, Toyokuni T, Wu L, Berk AJ, Cherry SR, Phelps ME and Herschman HR (1999). Repetitive, non-invasive imaging of the dopamine D2 receptor as a reporter gene in living animals. Gene Ther., 6, 785-791. McKenzie R, Fried MW, Sallie R, Conjeevaram H, Di Bisceglie AM, Park Y, Savarese B, Kleiner D, Tsokos M, Luciano C, Pruett T, Syotka JL, Straus SE and Hoofnagle JH (1995) Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B. N. Engl. J. Med., 333, 1099-1105. Misra HK, Knaus EE, Wiebe LI and Tyrell (1986) Synthesis of I31 I, I25 I, 123I, and 82Br labeled 5-halo-l-(2deoxy-2-fluoro-p-D-arabinofuranosyl)uracils. Appl. Radial, hot.. 37, 901-905. Misteli T and Spector D (1997) Applications of the green fluorecent protein in cell biology and biotechnology. Nature Biotech., 15, 961-964. Moerlein SM, Laufer P and Stocklin (1985) Synthesis of no-carrier-added radiobrominated N-alkylated analogues of spiperone. J. Labelled Compd. Radiopharm., 22, 1007-1022. Monclus M, Luxen A, Cool V, Damhaut P, Velu T and Goldman S (1997a) Development of a positron emission tomography radiopharmaceutical for imaging thymidine kinase gene expression: synthesis jo and in vitro evaluation of 9-{(3-[ F]fluoro-l-hydroxy-2-propoxy)methyl}guanine. Bioorg. Med. Chem.Lett.,7, 1879-82. Monclus M, Luxen A, Cool V, Damhaut P, Schurins A-L, Havet K, Baudson N, Van Naemen J, Velu T and 18 Goldman S (1997b) Synthesis of R- and (S)-9-{(3-[ F]fluoro-l-hydroxy-2-propoxy)methyl}guanine: Radiopharmaceutical for gene therapy. J. Labelled Compd. Radiopharm., 40, 20-22. Monclus M, Damhaut P, Luxen A, Velu T and Goldman S (1999a) In vitro validation of R- and (S)-9-(l1R [ F]fluoro-3-hydroxy-2-propoxy)methyl}guanine as radiopharmaceuticals for gene therapy. J. Labelled Compd. Radiopharm., 42, S627-629. Monclus M, Damhaut P, Kuhnast B, Tavitian B, Hantraye P, Velu T, Luxen A and Goldman S (1999b) In vivo validation in rat and monkey of rac-9-{(l-[ F]fluoro-3-hydroxy-2-propoxy)methyl}guanine: A radiopharmaceuticals for gene therapy. J. Labelled Compd. Radiopharm., 42, SI0-12. Morin KW, Atrazheva ED, Knaus EE and Wiebe LI (1997a) Synthesis and cellular uptake of 2'-substituted analogs of (E)-5-(2-[ I]iodovinyl)-2'-deoxyuridine in tumor cells transduced with the herpes simplex type-1 thymidine kinase gene. Evaluation as probes for monitoring gene therapy. J. Med. Chem., 40, 2184-2190. Morin KW, Knaus EE and Wiebe LI (1997b) Non-invasive scintigraphic monitoring of gene expression in a HSV-1 thymidine kinase gene therapy model. Nucl. Med. Comm., 18, 599-605. Namavari M, Barrio JR, Toyokuni T, Gambhir SS, Cherry SR, Herschman HR, Phelps ME and Satyamurthy 18 N (2000) Synthesis of 8-[ Fjfluoroguanine derivatives: In vivo probes for imaging gene expression with positron emission tomography. Nucl. Med. Biol., 27, 157-162. O'Neal WKR, Zhou H, Langston C, Rice K, Carey D and Beaudet AL (2000) Multiple advantages of afetoprotein as a marker for in vivo gene transfer. Mol. Ther., 2, 640-648. IQ

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Pan D, Gambhir SS, Toyokuni T, Iyer MR, Acharya N, Phelps ME and Barrio JR (1998) Rapid synthesis of a 5 -fluorinated oligodeoxynucleotide: a model antisense probe for use in imaging with positron emission tomography (PET). Bioorg. Med. Chem. Lett., 8, 1317-20. Pan D, Toyokuni T, Barrio JR, Satyamurthy N, Phelps ME and Gambhir SS (1999) Synthesis of a fluorine-18 labeled antisense oligodeoxynucleotide as a probe for imaging gene expression. J. A/we/. Med., 40, 82P. Pentlow KS, Graham MC, Lambrecht RM, Daghighian F, Bacharach SL, Bendriem B, Finn RD, Jordan K, Kalaigian H, Karp JS, Robeson WR and Larson SM (1996) Quantitative imaging of iodine-124 with PET. J. Nucl. Med., 37, 1557-1562. Phelps ME, Mazziotta JC and Schelbert HR (1986) Positron Emission Tomography and Autoradiography: Principles and Applications for the Brain and Heart, Raven Press, New York. Phelps ME (2000) PET: The merging of biology and imaging into molecular imaging. J. Nucl. Med., 41, 661 681 Phillips JA, Craig SJ, Bayley D, Christian RA, Geary R and Nicklin PL (1997) Pharmacokinetics, metabolism, and elimination of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A) after intravenous and subcutaneous administration. Biochem. Pharmacol., 54, 657-668. Rogers BE, Rosenfeld ME, Khazaeli MB, Mikheeva G, Stackhouse MA, Liu T, Curiel DT and Buchsbaum DJ (1997) Localization of iodine-^S-mlP-Des-Met^-bombesin (7-13)NH2 in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer. J. Nucl. Med., 38, 1221-1229. Saito Y, Price RW, Rottenberg DA, Fox JJ, Su T-L, Watanabe KA and Philips FS (1982) Quantitative autoradiographic mapping of herpes simplex virus encephalitis with radiolabeled antiviral drug. Science, 217, 1151-1153. Saito Y, Rubenstein R, Price R, Fox JJ and Watanabe KA (1984) Diagnostic imaging of herpes simplex virus encephalitis using antiviral drug: autoradiographic assessment in animal model. Ann. Neural., 15, 54858. Satyamurthy N, Bida GT, Barrio JR, Luxen A, Mazziotta JC, Huang SC and Phelps ME (1986) No-carrieradded 3-(2 -['8F]fluoroethyl)spiperone, a new dopamine receptor-binding tracer for positron emission tomography. Nucl. Med. Biol., 13, 617-624. Shiue C-Y, Fowler JS, Wolf AP, Watanabe M and Amett CD (1985) Synthesis and specific activity determinations of no-carrier-added (NCA) 18F-labeled butyrophenone neuroleptics-benperidoK haloperidol, spiroperidol and pipamperone. J. Nucl. Med., 26, 181-186. Shiue C-Y, Fowler JS, Wolf AP, McPherson DW, Amett CD and Zecca L (1986) No-carrier-added fluorine18-labeled N-methylspiroperidol: Synthesis and biodistribution in mice. J. Nucl. Med., 27, 226-234. Shiue C-Y, Bai L-Q, Teng R-R, Arnett CD and Wolf AP (1987) No-carrier-added N-(3[l8F]fluoropropyl)spiroperidol: Biodistribution in mice and tomographic studies in a baboon. J. Nucl. Med., 28, 1164–1170. Shiue C-Y, Hustinx R, Zhuang H, Shiue GG, Alavi AA and Eck SL (1999) 9-[(3-[ F]fluoro-l-hydroxy-218 propoxy)methyl]guanine ([ FjFHPG): A promising agent for monitoring HSV1-TK gene transfer to tumors. J. Labelled Compd. Radiopharm., 42, SI3–15. IQ

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as gene therapy imaging agents: A comparison. J. Nucl. Med., 41, 233P. Smith AE (1995) Viral vectors in gene therapy. Annu Rev Microbiol, 49, 807–838. Sokoloff L, Reivich M, Kennedy C, DesRosiers MH, Patlak CS, Pettigrew KD, Sakurada D and Shinohara M 14

(1977) The [ Cjdeoxyglucose method for the measurement of local cerebral glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. /. Neurochem,, 28, 897-916. Srinivasan A, Gambhir SS, Green AL, Cherry SR, Sharfstein S, Barrio JR, Satyamurthy N, Namavari M, Wu L, Berk AJ, Phelps ME and Herschman H (1996) A PET reporter gene/PET reporter probe technology for repeatedly imaging gene expression in living animals. J. Nucl. Med., 43, 107P. Tatum EL (1966) Molecular biology, nucleic acids and the future of medicine Perspect Biol. Med., 10, 19–32. Tavitian B, Terrazzino S, Kuhnast B, Marzabal S, Stettler O, Dolle F, Deverre J-R, Jobert A, Hinnen F, Bendriem B, Crouzel C and Giamberardino LD (1998) In vivo imaging of oligonucleotides with positron emission tomography. Nature Med., 4, 467–471. Tjuvajev JG, Stockhammer G, Desai R, Uehara H, Watanabe K, Gansbacher B and Blasberg RG (1995) imaging the expression of transfected genes in vivo. Cancer Res., 55, 6126–6132. Tjuvajev JG, Finn R, Watanabe K, Joshi R, Oku T, Kennedy J, Beattie B, Koutcher J, Larson S and Blasberg RG (1996) Non-invasive imaging of herpes virus thymidine kinase gene transfer and expression: a potential method for monitoring clinical gene therapy. Cancer Res., 56, 4087–4095. Tjuvajev JG, Avril N, Oku T, Sasajima T, Miyagawa T, Joshi R, Safer M, Beattie B, DiResta G, Daghighian F, Augensen F, Koutcher J, Zweit J, Humm J, Larson S, Finn R and Blasberg RG (1998) Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography. Cancer Res., 58, 4333–4341. Tjuvajev JG, Chen SH, Joshi A, Joshi R, Guo ZS, Balatoni J, Ballon D, Koutcher J, Finn R, Woo SL and Blasberg RG (1999) Imaging adenoviral-mediated herpes virus thymidine kinase gene transfer and expression in vivo. Cancer Res., 59, 5186–5193. Tovell DR, Samuel J, Mercer JR, Misra HK, Xu L, Wiebe LI, Tyrrell DL and Knaus EE (1988) The in vitro evaluation of nucleoside analogues as probes for use in the non-invasive diagnosis of herpes simplex encephalitis. Drug Des. Delivery, 3, 213–221. van Langevelde A, van der Molen HD, Journee-de Korver JG. Paans AM, Pauwels EK and Vaalburg W (1988) Potential radiopharmaceuticals for the detection of ocular melanoma. Part III. A study with i4C and UC labelled tyrosine and dihydroxyphenylalanine. Eur. J. Nucl. Med., 14, 382-387. Verma I'M and Somia N (1997) Gene therapy-Promise, problems and prospects. Nature, 389, 239-242. Wagner HN, Jr., Burns HD, Dannals RF, Wong DF, Langstrom B, Duelfer T, Frost JJ, Ravert HT, Links JM, Rosenbloom SB, Lukas SE, Kramer AV and Kuhar MJ(1983) Imaging dopamine receptors in the human brain by positron emission tomography. Science, 221, 1264-1266. Weissleder R, Simonova M, Bogdanova A, Bredow S, Enochs WS and Bogdanov A Jr (1997) MR imaging and scintigraphy of gene expression through melanin induction. Radiology, 204, 425-429.

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Welch MJ, Chi DY, Mathias CJ, Kilbourn MR, Brodack JW and Katzenellenbogan JA (1986) Biodistribution of N-alkyl and N-fluoroalkyl derivatives of spiroperidol: radiopharmaceuticals for PET studies of dopamine receptors. Nucl. Med. Biol., 13, 523-526. Wickstrom E (1986) Oligodeoxynucleotide stability in subcellular extracts and culture media J. Biochem. Biophys. Methods, 13,97-102. Wiebe LI, Morin KW and Knaus EE (1997) Radiopharmaceuticals to monitor gene transfer. Quart. J. Nucl. Med., 41, 79-89 Zinn KR, Buchsbaum DJ, Chaudhuri TR, Mountz JM, Grizzle WE and Rogers BE (2000) "mTc-P2045: A new peptide for imaging somatostatin receptor gene transfer. J. Nucl. Med., 41, 8IP. Zoltick PW and Wilson JM (2000) A quantitative nonimmunogenic transgene product for evaluating vectors in nonhuman primates. Mol. Ther., 2, 657-659.

16. MECHANISM OF TARGET SPECIFIC UPTAKE USING EXAMPLES OF MUSCARINIC RECEPTOR BINDING RADIOTRACERS WILLIAM C. ECKELMAN Warren Grant Magnuson Clinical Center, PET Department, 10 Center Drive, Bethesda, MD 20892

INTRODUCTION The mechanism of target specific uptake is related to the biochemical process involved since site directed radiotracers includes enzyme substrates, inhibitors and ligands that interact with transport proteins as well as membrane bound or cytosolic binding sites. More recently, investigators have included mRNA and DNA as specific targets. Fortunately, all of these processes are mathematically similar in the simplest case of the law of mass action for a ligand and a single binding site. The enzyme velocity, V equals Vmax[S]/([S] + Km) whereas specific binding, B equals Bmax[L]/([L] + KD). Motulsky notes a number of important differences(Motulsky, 1999). Receptor ligand interactions take from minutes to hours to reach equilibrium, whereas enzyme assays reach steady state (defined as a constant rate of product accumulation) in seconds. The specific binding equation is valid at equilibrium; the equation used to analyze enzyme kinetic data is valid when the rate of product formation is constant, so product accumulates at a constant rate. While KD is a equilibrium dissociation constant, Km is not a binding constant since its value includes the affinity of substrate for enzyme and the kinetics by which the substrate bound to the enzyme is converted to product. The use of radioligands to measure specific target uptake in vitro has been available since Jensen and Jacobson developed [3H]estradiol in the early 1960's (Jensen & Jacobson, 1962 ). Most of the early studies were limited by the low specific activity and low affinity of the radiolabel. The history of radioligands for the muscarinic system demonstrates the evolution of radiotracers. The muscarinic acetylcholine receptor (mAChR) was one of the earliest receptors studied with in vitro techniques. As early as 1973, Farrow & O'Brien(Farrow and O'Brien, 1973) described the use of [3H]atropine to define the mAChR. A year later, Held and Ariens (Beld & Ariens, 1974) reported the use of stereospecific binding of benzetimide using the (+) and (-) isomers. They reported that [3H]atropine had a specific activity of 0.4 Ci/mmol and [3H]benzetimide had a specific activity of 0.6 Ci/mmol. This can be compared with the value of 30 Ci/mmol for one tritium per molecule. They were able to show specific binding for (+) benzetimide and atropine, but not for (-) benzetimide. This was the first demonstration in the muscarinic receptor field of stereospecific binding. With the development of higher affinity and higher specific activity radioligands such as [3H]quinuclidinyl benzilate (QNB), receptor distribution was mapped in isolated tissue(Yamamura et al., 1974). The first ligand to map mAChR in humans in vivo was the radioiodinated form of QNB, 3-R-quinuclidinyl 4-S-iodobenzilate (RS IQNB)(Eckelman et al., 1984). This was, in fact, the first molecular assay of mammalian neuroreceptor biology in a living subject. The use of receptor-binding radiotracers for

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external brain imaging differs from their use in vitro. In order for a radiotracer to be useful in vivo, its distribution must be driven by the local receptor concentration rather than by local blood flow or membrane transport properties, so that the images obtained primarily reflect receptor binding. A comprehensive kinetic analysis of RS IQNB in rats was performed by Sawada et al.(Sawada et al., 1990). It showed that the uptake in cerebrum is essentially irreversible during the first 360 min after intravenous administration and that the rate of RS IQNB tissue uptake depends on transport across the blood-brain barrier (BBB) as well as on the rate of binding to the receptor. However, the later data (-24 hours) are sensitive to receptor concentration. Clinical studies with RS IQNB have indicated that it is responsive to changes in receptor concentration at late times post-injection (Weinberger et al., 1991; Weinberger et al., 1992). However, this ligand does not demonstrate significant muscarinic subtype selectivity. This illustrates a key point in the development of receptor binding radiotracers. Twenty years ago, pharmaceuticals did not have high enough affinities, in light of the receptor concentration, to produce high target to nontarget ratios. Therefore, the design goal was to produce radiotracers with high affinity (< 1 nM). However, the distribution of these high affinity compounds was flow dependent and the influence of the receptor concentration was only realized at delayed times after injection, if at all. RS IQNB is an example of such a compound whose early distribution is flow dependent, but at 18 hours after injection the distribution is influenced by receptor concentration. Now, radiotracers with a range of binding potentials, (Bmax/KD), are being developed ranging from those that are notably affected by endogenous ligands (reversible ligands) to those that are flow or transport dependent (irreversible Hgands). Although generalizations are difficult to make for the use of these complicated ligands in vivo, the reversible ligands have KD values of ~2-5 nM and the irreversible ligands have KD values of « 1 nM for target receptors present in ~10-100 nM concentration. STEPS IN THE DEVELOPMENT OF TARGET SPECIFIC RADIOLIGANDS. We have proposed several paradigms outlining the steps necessary to develop a new target specific radioligand (Eckelman et al., 1979; Eckelman 1982; Eckelman 1985a; Eckelman 1985b; Eckelman 1986; Eckelman et al., 1992). One such list contains multiple steps to develop an imaging agent. 1. The choice of a saturable binding site. This choice has traditionally been made on the basis of autopsy data that show a change in saturable binding site as a function of disease or more indirectly based on the current class of drugs used to treat a specific disease. However, with the advent of genetic linkage studies and genetic screening, a new avenue of radiotracer development has been developed. For example, the current state of molecular genetics of Alzheimer's disease (AD) focuses on four genes: the p-amyloid precursor protein (P-APP) gene, the presenilin 1 gene and the presenilin 2 gene (St. George-Hyslop, 2000). In addition, the e4 allele of apolipoprotein E (APOE) gene is associated with an increased risk for late-onset AD. Approximately 30-50% of the population risk for AD can be attributed to genetic factors with a final risk or 38% by age of 85 years. The characteristic neuropathology is neurofibrillary tangles and amyloid plaques. The result of the phenotypic changes is a result of an alteration in the processing of p-APP favoring the production of the potentially toxic Ap42 protein. Few of the imaging studies to date have been based on these gene products, although binding to AP42 protein has been reported (Skovronsky et al., 2000). Although this is an indirect measure of the phenotype, [I8F]FDG has been used to identify a sub-population of AD patients with the APOE gene (Kuhl et al. 1999). The authors

MECHANISM OF TARGET SPECIFIC UPTAKE found changes in FDG distribution before clinical manifestations. However, the majority of the effort in radiopharmaceutical development has been to design ligands based on deficits found at autopsy studies of AD patients. The previous focus has been on the development of M2 subtype selective cholinergic ligands, based on the observation that this subtype is lost in the cerebral cortex in Alzheimer's disease (Quirion et al., 1989; Aubert et al., 1992; Rodriguez-Puertas et al., 1997). 2. Use of a mathematical model to choose potential receptor-specific radiopharmaceuticals. The estimation of the Krj and receptor concentration (Bmax) for the target compound and the maximal bound to free ratio (B/F). Having chosen a receptor system based on a disease state, the use of a mathematical model to choose potential receptor-specific radiopharmaceuticals is key. A simple model has been put forth by Katzenellenbogen (Katzenellenbogen et al., 1982) and Eckelman (Eckelman et al., 1979) as a first approximation. At high specific activity, the maximal B/F ratio will be Bmax/KD. Certainly, distribution factors, protein binding, metabolism, and other interactions will decrease the maximal B/F ratio. Therefore, this criterion is necessary but not sufficient. This estimation is especially important, as targeting of receptor systems with pM concentration becomes more prevalent. 3. Use of •*H-labeled compounds to determine distribution in vivo. With the number of ^H-labeled compounds now available commercially, the use of ^H-labeled compounds is an efficient method to determine if a particular binding site ligand is suitable for radiolabeling with a gamma emitting radionuclide. At the maximum specific activity of 30 Ci/mmol for one tritium per molecule, the specific activity is sufficient to produce the maximum B/F ratio in cases where the binding site concentration is ~ nM. The exact analog (fluorinated derivative and, especially, a technetium derivative) is usually not available in the tritiated form and that has to be taken into account. One can expect a weaker KD with large perturbations in the structure of the parent compound. 4. The preparation of the non-radioactive analog and determination of in vitro and in vivo stability. The preparation of the non-radioactive substituted ligand is often the next step. This avoids the problems with no-carrier-added synthesis until the substituted ligand is shown to be a true tracer for the unsubstituted receptor binding ligand, especially in those cases where the substitution of the radionuclide introduces a substantial perturbation. This also produces the necessary reference compounds for the radiolabeled compound. The preparation of the non-radioactive compound better defines the radioactive compound because classical analytical methods can be used, whereas with the high specific activity compound, chromatography is the only analytical tool usually available for identification and quantification. In the current regulatory climate, the nonradioactive compound is also needed to carry out the necessary toxicology and pathology studies. Most compounds are either chemically unstable and/or metabolized in vivo. Therefore, the stability of the new derivative should be determined in plasma and in liver. The most convenient model for the enzymatic activity of the liver is commercially available cryopreserved hepatocytes. One difficulty with using the non-radioactive compound for stability testing is caused by the large

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HANDBOOK OF RADIOPHARMACEUTICALS concentrations used, which will most likely result in a second order reaction whereas the high specific activity ligand will most likely be in a pseudo first order reaction. This problem has been solved with the use of a combination liquid chromatography/ mass spectrometry (LC/MS) analysis where picograms of material can often be analyzed. For example, the metabolic profile of the selective M2 muscarinic agonist, fluorine-18 labeled 3-(3-((3-fluoropropyl)thio)-l,2,5-thiadiazol4-yl)-l,2,5,6-tetrahydro-l-methylpyridine (FP-TZTP) was studied in rat and human hepatocytes using LC/MS/MS with non-radioactive FP-TZTP and carrier-added [18F]FP-TZTP(Ma et a/., In Press). Using non-radioactive FP-TZTP in human and rat hepatocytes, the major metabolite resulted from oxidation of the nitrogen in the 1-methyltetrahydropyridine ring. Other metabolites result from sulfur-oxidation, demethylation of the tertiary amine, and oxidation of the tetrahydropyridine ring. Rat plasma was also analyzed after intravenous injection of carrier added [18F]FP-TZTP. The HPLC with both a radioactivity and mass spectrometric detector indicated that the metabolism profile in vivo is similar to that obtained in vitro. The N-oxidation product was the major metabolite. The major radioactivity peak in brain extract is the parent [I8F]FP-TZTP, although there is less than 5% parent in plasma in 30 min. From the knowledge of the structure of the metabolites, a two step extraction sequence that allows the isolation of unmetabolized parent compound was developed. In summary, there are two advantages of using LC/MS to define the metabolites of a potential radiopharmaceutical: (1) the identity of the metabolites are known and therefore questions about the presence of metabolites in the target tissue can be answered and (2) simple extraction procedures can be developed to determine the percentage of parent in the plasma. Such a method allows rapid determination of the parent fraction in plasma and does not require time-consuming chromatographic analysis.

5. The evaluation of various physical parameters of the non-radioactive derivative (StructureDistribution Relationship, SDR). Just as structure-activity relationships have been the foundation of classic drug design, structure-distribution studies are the backbone of radiopharmaceutical development. The use of both theoretical and experimental parameters can direct the choice of radioligand. Many of the earlier radiopharmaceuticals were water-soluble, polar compounds that are excreted by the kidneys. As radioligands were developed that cross cell membranes, the properties of these compounds were often correlated with a particular physicochemical property. Most often, the property is lipophilicity determined by either measuring the organic-aqueous partition coefficient or using a related technique such as reversed phase high-pressure liquid chromatography. Various organic solvents have been used to determine the partition coefficient. The goal is to choose an organic solvent that most closely models the cell membrane. Franks and Lieb published the classic example of the relationship between the activity of general anesthetics and the partition coefficient. (Franks & Lieb, 1978). Various solvents were used including vegetable oil, n-hexadecane and n-octanol. The vegetable oil system did not give a good correlation when hydroxy-containing anesthetics were included, and the hexadecane system was poor for most polar anesthetics. Octanol-water on the other hand, produced a good correlation for a broad range of anesthetics. The authors concluded that octanol best represents the cell membrane involved in anesthesia. Oftentimes, a poor correlation is due to the dependence on

MECHANISM OF TARGET SPECIFIC UPTAKE diffusion phenomena rather than an incorrect choice of solvent (Stein, 1986). The net flux across a cell membrane is directly proportional to the diffusion coefficient and the partition coefficient of the ligand and indirectly proportional to the thickness of the membrane. The combination of these three parameters is the permeability coefficient. If diffusion is an important parameter in the cell membrane transport, then it must be taken into account. Derivations of the size-corrected permeability coefficient then become an important part of the evaluation. These can be calculated by measuring the permeability coefficient-partition coefficient ratio as a function of the molecular volume. Since the molecular volume is difficult to obtain for anything but the simplest organic compounds, a function of the molecular weight (MW), usually the square root, is often used. In the radiopharmaceutical context, the percentage dose/g tissue gives a value proportional to the permeability coefficient. The dependence on molecular weight can be determined and then the molecular weight independent percentage dose/g can be plotted against the partition coefficient to determine the best linear model. This is more sophisticated than the model by Fenstermacher and Rapoport (Fenstermacher & Rapoport, 1984) because it determines the molecular volume dependence for each membrane. It is more difficult because the molecule volume must be estimated. 6. In vivo displacement of the ^H or 125I labeled compound with the non-radioactive derivative. If a tritium labeled compound is available for the binding site in question, the ability of the nonradioactive derivative to compete for the receptor should predict the distribution of the radioactive form of the same compound. For example, a series of halogenated derivatives of the muscarinic receptor binding ligand, 3-quinuclidinyl benzilate, have been synthesized and tested for their ability to compete with ^H-QNB for the muscarinic receptor. The logit plot of the displacement of ^H-QNB by each of the analogues was constructed (Eckelman et al., 1984). This approach compares the ratio of the percentage dose/g bound in the brain in the presence and absence of 50 nrnol of non-radioactive analogue per animal to the relative binding as measured in the in vitro radioreceptor assay. The single compound that is less potent than predicted is 3-quinuclidinyl-4~ iodobenzilate. Most groups today proceed with radiolabeling each analog and comparing the SDR using radiolabeled ligands as opposed to using this approach in spite of the obvious efficiencies. In another example of the efficiency of testing a series of analogues, subtype selectivity for the muscarinic acetylcholine receptor (mAChR) was determined using a single radiolabeled probe and various nonlabeled analogues. In this particular case, the intent was to choose an analog of QNB, which would be M2 subtype specific and easily radiolabeled with the positron emitting radionuclide F-18(Lee et al., 1995). Fluorine-19 labeled alkyl analogues of quinuclidinyl benzilate (QNB) were synthesized by stereoselective reactions. In vivo competitive binding studies were performed in rat brain using (R)-3-quinuclidinyl (S)-4-[125I]iodobenzilate (IQNB). Five, 50, or 500 nmol of the non-radioactive ligands were coinjected intravenously in rat with 8 prnol of the radioligand. Cold (R,R)-IQNB blocked (R,R)-[125I]IQNB in a dose-dependent manner, without showing regional specificity. For the (R,S)-fluoromethyl, fluoroethyl, and fluoropropyl derivatives, a higher percent blockade was seen at 20 and 200 nmol/Kg in M2 predominant

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HANDBOOK OF RADIOPHARMACEUTICALS tissues (medulla, pons, and cerebellum) than in Ml predominant tissues (cortex, striatum and hippocampus). The blockade pattern of the radioligand also correlated qualitatively with the percentage of M2 receptors in the region. The S-quinuclidinyl analogues showed M2 selectivity but less efficient blockade of the radioligand, indicating lower affinities. Radioligand bound to the medulla was inversely correlated to the M2 relative binding affinity of the fluoroalkyl analogues. These results indicate that the non-radioactive ligand blocks the radioligand based on the affinity of the non-radioactive ligand for a particular receptor subtype compared to the affinity of the radioligand for the same receptor subtype. Of the seven compounds evaluated, (R,S)fluoromethyl-QNB appears to show the most selectivity for the M2 subtype in competition studies in vivo. Previously, the non-radioactive (R,R) diastereomer was shown to display an 8-fold selectivity for Ml over M2 in vitro while the non-radioactive (R,S) diastereomer displayed a 7-fold selectivity for M2 over Ml. As a result, both the (R,S)-fluoromethyl-QNB and the (R,R)-fluoromethyl-QNB were radiolabeled with F-18. In the rat, uptake of (R,S)-[l8F]FMeQNB was nearly uniform in all brain regions mirroring the concentration of M2 subtype. The uptake was reduced by 36-54% in all brain regions upon coinjection with 200 nmol/Kg of unlabeled ligand. An injection of (R,S)['8F]FMeQNB followed at 60 min by injection of unlabeled ligand and subsequent sacrifice at 120 min displaced 30 to 50 % of radioactivity in the pons, medulla, and cerebellum, which contain a high proportion of M2 subtype. The most dramatic displacement and blocking of (R,S)[18F]FMeQNB was observed in the heart. In rhesus monkey, the compound showed prolonged uptake and retention in the brain. In the blood, the parent compound degraded rapidly to a single radiolabeled polar metabolite believed to be fluoride. Within 30 min the parent compound represented less than 5% of the plasma activity. Displacement with (R)-QNB was generally slow, but was more rapid from those tissues, which contain a higher proportion of M2 subtype. The results are consistent with the hypothesis that (R,S)-[18F]FMeQNB is M2 selective in vivo. The value of screening a number of compounds in the non-radioactive form is especially evident for fluorinated compounds where the synthesis of the non-radioactive compound is often more straightforward than the synthesis of the radioactive fluorinated compound. On the other hand, (R,R)-[I8F]FMeQNB showed higher uptake in those brain regions containing a higher concentration of Ml subtype. Uptake in the heart at 60 min was much lower than that observed with the R,S diastereomer. Blocking with unlabeled (R.S)-FMeQNB is only significant in the heart, thalamus, and pons. Blocking with unlabeled (R.R)-FMeQNB is quite uniform in all brain regions. Displacement with (R)-QNB shows a more varying amount displaced. These results are consistent with (R,R)-['8F]FMeQNB being Ml selective in vivo.

7. Preparation of the radioactive derivative and use of preinjection, coinjection, or postinjection to decrease effective specific activity of the radioactive derivative. Animal distribution studies using the radiolabeled ligand are the most telling experiments. Besides the requirement that the radioactivity be present in the target organ with a target to nontarget ratio of ~2 for PET and

MECHANISM OF TARGET SPECIFIC UPTAKE higher for SPECT and planar imaging, radioligands for saturable binding sites must also show specific binding. In general, this is carried out by using pre-, co- or post-injection of a known receptor binding biochemical or drug. Since the definition of a receptor and a receptor binding radiotracer is operational, the distribution of the radioligand must fit the criteria of high affinity, specificity including stereospecificity, saturability, and correlation with biological activity. There are situations where these criteria cannot be tested. This inability to test these criteria occurs if the distribution of the binding site is homogeneous throughout the gray matter or if there are no specific ligands for that binding site from a different chemical class. One example of this is the putative M2 subtype specific ligand, [18F]FP-TZTP (Kiesewetter et al., 1995). Autoradiography using no-carrier-added [i8F]FP-TZTP confirmed the uniform distribution of radioactivity characteristic of the M2 pattern of distribution as described with immunocytochemical localization studies (Levey, 1993). At 1 h after injection, co-injection of P-TZTP at 20, 200 and 2000 nmol/Kg inhibited [18F]FP-TZTP uptake in a dose dependent manner. The difference in brain regions between each dose level was significant, except for the 20 nmol/Kg value in medulla. The brain distribution of the agonist [18F]FP-TZTP was unaffected by co-injection of M2 selective antagonists. At 5 minutes after IV injection of [I8F]FP-TZTP, the uptake in the heart was inhibited 55% with a co-injection of P-TZTP, which would suggest M2 selectivity. There were no M2 subtype selective agonists or antagonists of a different chemical class that blocked the binding of [i8F]FP-TZTP. PET studies in isoflurane-anesthetized rhesus monkeys were performed to assess the in vivo behavior of [!8F]FP-TZTP(Carson et al, 1998 ). The volume of distribution (V) representing total tracer binding, i.e., free, nonspecifically bound, and specifically bound, were similar in cortical regions, basal ganglia, and thalamus, but were significantly lower in the cerebellum which agreed with the receptor distribution reported for rat and monkey (Li et al., 1991; Flynn et al., 1993; Yamamura et al., 1974). Pre-administering 200-400 nmol/kg of nonradioactive FP-TZTP produced a dramatic reduction in total binding of ~50% in cerebellum and 60-70% in other gray matter regions. Similar blockade was seen in analogous rat studies (Kiesewetter et al., 1995). In addition to pre-blocking studies, displacement of [18F]FP-TZTP with 80 nmol/kg of FP-TZTP at 45 min post injection caused a distinct increase in net efflux with decreases of 20%, 36%, and 41% in cerebellum, cortex, and thalamus, respectively(Kiesewetter et al., 1999). The sensitivity of [!8F]FP-TZTP binding to changes in brain acetylcholine was assessed by administering physostigmine, an acetylcholinesterase inhibitor, by i.v. infusion beginning 30 min before tracer injection. Physostigmine produced a 35% reduction in cortical specific binding, consistent with increased competition from acetylcholine (Carson et al., 1998), Although these data were consistent with FP-TZTP binding to the M2 receptor subtype, they were not conclusive in proving M2 selective binding. Unfortunately, no subtype specific agonists with high affinity that cross the blood-brain barrier have been identified so conventional in vivo muscarinic receptor blocking studies with high affinity compounds of different chemical structures were not possible. One solution to this problem of not being able to use the traditional operational definition of a receptor binding radiotracer is the use of gene-altered mice. In the example of [18F]FP-TZTP, the development of both M2 and M4 knockout mice (Gomeza et al., 1999a; Gomeza et al., 1999b)

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made possible the study of [18F]FP-TZTP in these knockout models to further elucidate the mechanism of localization of this radiotracer. Using ex vivo autoradiography, regional brain localization of [18F]FP-TZTP in M2 knockout (KO) vs wild type (WT) mice was determined. The decrease in [18F]FP-TZTP uptake in M2 KO mouse brain regions (amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, inferior colliculus, superior colliculus, and thalamus) at 30 min after IV injection was relatively similar to that found in the ST mice, ranging from 51.3 to 61.4%. While a significant decrease (p<0.01) of [18F]FP-TZTP uptake was observed in all brain regions examined for the M2 KO vs WT, similar studies with M4 KO vs WT exhibited no significant decreases in all brain regions. In conclusion, the pharmacologic data, which suggested that [18F]FP-TZTP is binding to the M2 receptor, has been confirmed by these M2 KO mice studies. The use of KO mice is a powerful tool to determine subtype selectivity of potential receptor binding radiotracers (Jagoda et al., 2001). Use of active and inactive stereomers of the radioactive analog. The use of an active and inactive stereomers is especially important in human studies where injecting high concentrations of nonradioactive compound either pre-co or post-injection of the radioligand could cause pharmacologic effects. The active and inactive stereomers can both be injected at high specific activity thereby avoiding that problem. The key to using the stereomer pair is to show that indeed the "inactive" isomer represents the free and nonspecific pools only and has the same metabolic profile. QNB, the nonselective muscarinic antagonist, has one chiral center in the quinuclidinyl moiety and, when substituted in one of the two benzylic acid phenyl groups, has another chiral center at the benzylic carbonyl carbon atom. This creates the possibility of four isomers that are identified by designating the stereochemistry of the quinuclidinyl center first and the benzylic center second (Kiesewetter et al., 1994). We have compared R,R-[123I]IQNB and S,S-[I23I]IQNB in four volunteers to determine if the S,S enantiomer can be used as an indication of nonspecific binding (NSB) in the brain and the parotid gland (Hiramatsu et al., 1995). In all patients, metabolitecorrected plasma (MCP;%ID/g) clearance of the S,S IQNB was faster than that obtained with R,R IQNB. The plateau plasma value for both enantiomers was between 0.0003-0.001 %ID/g. The ratio of tissue concentration (T; %ID/sq.cm) to MCP obtained from planar imaging and blood sampling of four normal volunteers at 20-23 hours after injection were :

Kl (RR/SS) k2 (RR/SS) k3 (RR/SS) k4 (RR/SS) app. Vd (RR) app. Vd (SS)

Cortex (Ml) 0.56 1.9 4.5 0.61 52.8 37.9

Cerebellum (M2) 0.61 2.0 1.4 0.16 16.7 28.7

Parotid(M3) 0.94 2.9 14.6 0.71 21.0 33.0

MECHANISM OF TARGET SPECIFIC UPTAKE The T/MCP ratio for R,R-IQNB vs. S,S-IQNB is in agreement with the binding constants obtained in vitro [Kd (RR/SS) in nM] for Ml (0.34/4.6), for M2(4.2/9.0) and for M3 (8.1/40). Because of the difference in units between T and MCP, the high values for S,S IQNB can be due to either nonspecific binding or specific binding. In summary, our overall results showed that SS- and RRIQNB displayed stereospecific behavior with respect to binding to protein, metabolism, and tissue transport. However, these data suggest that S,S IQNB is not useful as a control for nonspecific binding. 9. Measurement of sensitivity of the radioligand, most often in non-human primates. Kinetic sensitivity has been defined as the ability of a physiochemical parameter to alter the time-activity data of a radio-indicator(Vera et al., 1989). In the context of radiotracers for high affinity sites, the time-activity curve in the target organ should be sensitive to a change in binding site concentration. In order to use the more practical single-scan technique (Carson, 1996), the change must be recorded at a single time point. This information is often normalized to the metabolitecorrected plasma concentration of the radiotracer or a reference organ shown to be equivalent, and either the original target organ concentration or these target to non-target ratios are compared to the receptor concentration obtained by analyzing tissue samples in vitro. A 1991 Nuclear Medicine and Biology editorial stated that "There is a pressing need to study positron emittingand 123I-labeled single-photon emitting-radiotracers to determine their sensitivity to biochemical changes and match that with diseases that undergo a similar change. This step has been delayed by the incessant development of new ligands that bind specifically in vivo, but are validated no further. This proof of specific binding is a necessary but by no means sufficient step in the complete validation of a probe for a biochemical process. Although the increased sensitivity of PET and SPECT imaging devices and the rapid development of specific biochemical radiotracers is encouraging, only with increased attention to the validation of these biochemical radiotracers will the clinical importance of SPECT and PET be realized" (Eckelman, 1998). One of the first biochemical probes, 2-[^F] fluoro-2-deoxyglucose, set a high standard for the development of other probes to be used in vivo. Using * ^C-deoxyglucose in studies spanning three decades, Dr. Louis Sokoloff developed a method of measuring glucose utilization in differing behavioral states, seizures, during development, during sensory stimulation, and following administration of drugs. This brilliant integration of science started with the methods for the measurement of cerebral blood flow developed by Seymour Kety, including the quantitative autoradiographic procedure using inert diffusible tracers for measuring local metabolic rates. This laid the groundwork for the crucial development: the determination of glucose utilization as a measure of energy metabolism and functional activity. Since the use of radiolabeled glucose necessitated very short measurement times, the metabolically trapped 2-deoxyglucose was studied. This substrate, like glucose, was phosphorylated by hexokinase, but the product could not be converted to fructose-6-phosphate, the next step in the glycolytic pathway. 2-deoxyglucose-6phosphate accumulates in brain to high levels because it is a poor substrate for enzymes present in brain and because glucose-6-phosphatase activity is very low in brain. Thus, 2-deoxyglucose

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HANDBOOK OF RADIOPHARMACEUTICALS could be used as a tracer for glucose with the autoradiographic technique that had been devised for the local cerebral blood flow method. Drawing on his experience with enzyme kinetics, Sokoloff developed a quantitative model to measure the local glucose utilization. As a result new relationships were revealed and the full constellation and degree of participation of structures simultaneously activated or inhibited as a result of a given behavior or response to stimulation were demonstrated for the first time. Shortly thereafter, the method was adapted for use in human subjects and has become the most important technique for measuring brain function by external detection using PET. Numerous examples of radioligands that are specific and sensitive are presented in accompanying chapters in this book.

CONCLUSION The development of radioligands to monitor changes in muscarinic receptor density as a function of disease serves as a useful example of the approach to producing target specific uptake in vivo. As a result of these developmental efforts, [I8F]FP-TZTP has been developed. It is subtype specific, which is very important in the ever evolving developments in molecular biology. It is a radiolabeled agonist and, therefore, is theoretically capable of measuring not only receptor number but also second messenger interactions. Although the properties of [18F]FP-TZTP cannot be validated by the traditional operational definition of a receptor binding radiotracer, with the advent of genetic manipulation in mice, including the deletion of specific genes, the subtype specificity can be shown conclusively using muscarinic receptor knock-out mice. Finally, [18F]FP-TZTP is a reversible ligand and, therefore, can measure changes in endogenous neurotransmitter. [18F]FP-TZTP represents an exquisite example of the modem trend in radioligand design.

MECHANISM OF TARGET SPECIFIC UPTAKE ACKNOWLEDGMENT Those that have been involved include: BAUM BJ BLASBERG R BOULAY S.F. BRAUN A BUDINGER T CARRASQUILLO JA CARSON R.E. COHEN VI CONKLIN JJ DER M.G. DiROCCO RJ ENDRES C.J. ENGR FRANCIS BE

in the development of muscarinic radioligands reviewed in this manuscript GIBSON RE GOMEZA J GRISSOM M HERSCOVITCH P. HILL T HIRAGA S HIRAMATSU Y HOLMAN BL HUANG BX ITOK JAGODA E JURISSON SS KAPPOH KIESEWETTER DO

KURRASCH RHM LANGL LARSON S LEEJT MAY MACYNSKI AA MILETICH RS NANJAPPAN P NEUMANN RD NOWOTNIK DP NUNN AD OWENS E PAIK CH PARK SG

PATLAK C PETTIGREW K PIRRO J RAYEQ M.R. REBA RC ROSENSPIRE KC RZESZOTARSKI WJ SAWADA Y SHIMOJI K SIEMION J SOOD V.K. VALDEZIH WESSJ ZEEBERG B.R.

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REFERENCES Aubert I, Araujo DM, Cecyre D, Robitaille Y, Gauthier S and Quirion R (1992) Comparative alterations of nicotinic and muscarinic binding sites in Alzheimer's and Parkinson's diseases. J. Neurochem. 58: 529541. Beld AJ and Aliens EJ (1974) Stereospecific binding as a tool in attempts to localize and isolate muscarinic receptors. Part II. Binding of (plus)-benzetimide, (minus)- benzetimide and atropine to a fraction from bovine tracheal smooth muscle and to bovine caudate nucleus. Eur. J. Pharmacol. 25(2): 203-9. Carson RE (1996) Mathematical modeling and compartmental analysis. In Nuclear Medicine: Diagnosis and Therapy. 3rd Edition. Harbert J, Neumann R and Eckelman W. (eds), Thieme Press., New York, pp. 167–194. Carson RE, Kiesewetter DO, Jagoda E, Der MG, Herscovitch P and Eckelman WC (1998) Muscarinic cholinergic receptor measurements with [I8F]FP- TZTP: Control and competition studies. J. Cereb. Blood Flow & Metab. 18(10): 1130–1142. Eckelman WC (1982) In Receptor Binding Radiotracers. Boca Raton, FL, CRC Press, Inc. Eckelman WC (1985a) The design of cholinergic tracers in neurosciences. In Discussions in Neuro Sciences. Feindel WF. Gadian, RSJ, Magistretti D. , PL. and Zalutsky MR (eds)Netherlands, Foundation for the Study of the Nervous System. 11: 60–71. Eckelman WC (1985b) Potentials of receptor binding radiotracers. In New Brain Imaging Techniques in Cerebrovascular Diseases. Cahn J Lassen N. Paris, John Libbey Euro Text. 2: 113V124. Eckelman WC (1986) Receptor binding radiotracers, Takeda Science Foundation Symposium on Bioscience. Biomedical Imaging. Hayaishi O Torizuka K. Tokyo, Japan, Academic Press, Inc.: 357–374. Eckelman WC (1998) Sensitivity of new radiopharmaceuticals. Nucl. Med. Biol. 25(3): 169–173. Eckelman WC and Gibson RE (1992) The design of site directed radiopharmaceuticals for use in drug discovery. Boston MA., Birkhauser Boston, Inc. Eckelman WC, Gibson RE, Rzeszotarski WJ, Vieras F, Mazaitis JK, Francis B and Reba RC (1979) The design of receptor binding radiotracers. In Principles of Radiopharmacology. Colombetti L. (ed) CRC Press, New York. 1: 251–274. Eckelman WC, Grissom M, Conklin J, Rzeszotarski WJ, Gibson RE, Francis BE, Jagoda EM, Eng R and Reba RC (1984) In vivo competition studies with analogues of quinucidinyl benzilate. J. Pharm. Sci., 73, 529–533. Eckelman WC, Reba RC, Gibson RE, Rzeszotarski WJ, Vieras F, Mazaitis JK and Francis B (1979) Receptor binding radiotracers: a class of potential radiopharmaceuticals. J. Nucl. Med., 20, 350–357. Farrow JT and O'Brien RD (1973) Binding of atropine and muscarone to rat brain fractions and its relation to the acetylcholine receptor. Molecular Pharmacol., 9(1), 33–40. Fenstermacher J and Rapoport S. (1984) Blood-brain barrrier. In Handbook of Physiology, the Cardiovascular System IV. Renkin E Michel C. Bethesda, MD, American Physiological Society. Chapter 21:969–1000. Flynn DD and Mash DC (1993) Distinct kinetic binding properties of N-[3H]-methylscopolamine afford differential labeling and localization of Ml, M2, and M3 muscarinic receptor subtypes in primate brain. Synapse 14(4), 283–96. Franks NP and Lieb WR (1978). Where do general anesthetics act? Nature 274(5669): 339-42.

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Gomeza J, Shannon H, Kostenis E, Felder C, Zhang L, Brodkin J, Grinberg A, Sheng H and Wess J (1999a). Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl. Acad, Sci. USA, 96(4), 1692–7. Gomeza J, Zhang L, Kostenis E, Felder C, Bymaster F, Brodkin J, Shannon H, Xia B, Deng C and Wess J (1999b) Enhancement of Dl dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice. Proc Natl. Acad. Sci. USA, 96(18), 10483–8. Hiramatsu Y, Eckelman WC, Carrasquillo JA, Miletich RS, Valdez IH, Kurrasch RH, Macynski AA, Paik CH, Neumann RD and Baum BJ (1995) Kinetic analysis of muscarinic receptors in human brain and salivary gland in vivo. Am. J. Physiol, 268(6 Pt 2): Rl491–1499. Jagoda E, Kiesewetter DO, Shimoki K, Gomeza J, Wess J and Eckelman WC (2001) Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is decreased in M2 knockout but not in M4 knockout mice. Life Sciences, 68, 2634-. Jensen E and Jacobson H (1962) Basic guides to the mechanism of estrogen action. Recent Prog. Harm. Research 18, 387. ZKatzenellenbogen JA, Heiman DF, Carlson KE and Lloyd JE (1982) In vitro and in vivo steroid receptor assays in the design of estrogen radiopharmaceuticals. In Receptor Binding Radiotracers. Eckelman WC (ed) CRC Press, Boca Raton, Fl., 1: 93–126. Kiesewetter DO, Carson RE, Jagoda EM, Herscovitch P and Eckelman WC (1999) In vivo muscarinic binding of 3-(alkylthio)-3-thiadiazolyl tetrahydropyridines. Synapse, 31(1): 29–40. Kiesewetter DO, Lee J, Lang L, Park SG, Paik CH and Eckelman WC (1995) Preparation of 18F-labeled muscarinic agonist with M2 selectivity. J. Med. Chem,. 38(1), 5–8. Kiesewetter DO, Silverton JV and Eckelman WC (1994) Stereoselective synthesis of [R,R]IQNB and fluoroalkyl analogs of QNB. J. Labelled Compd. Radiopharm., 35, 419-421. Kuhl DE, Koeppe RA, Minoshima S, Snyder SE, Ficaro EP, Foster NL, Frey KA and Kilbourn MR (1999) In vivo mapping of cerebral acetylcholinesterase activity in aging and Alzheimer's disease [see comments]. Neurology, 52(4), 691–9. Lee JT, Paik CH, Kiesewetter DO, Park SG and Eckelman WC (1995) Evaluation of steroisomers of 4fluoroalkyl analogs of 3-quinuclidinyl benzilate in in vivo competition study for muscarinic receptor subtypes, the Ml, M2, and M3. Nuci Med. BioL, 22(6), 773–782. Levey Al (1993) Immunological localizaiton of ml-m5 muscarinic acetylcholine receptors in peripheral tissues and brain. Life Science 52(5-6): 441–448. Li M, YasudaRP, Wall SJ, Wellstein A and Wolfe BB (1991) Distribution of M2 muscarinic receptors in ratbrain using antisera selective for M2 receptors. Molecular Pharmacology, 40(1), 28–35. Ma Y, Kiesewetter DO, Jagoda E, Huang BX and Eckelman WC (In Press). Identification of metabolites of fluorine-1.8 labeled M2 muscarinic receptor agonist, FP-TZTP, produced by human and rat hepatocytes. J. Chromatography B. Motulsky H (1999) Analyzing data with GraphPad Prism. GraphPad Software Inc., San Diego, CA: p. 333. Quirion R? Aubert I, Labchak PA, Schaum RP, Teolis S, Gauthier S and Araujo DM (1989) Muscarinic receptor subtypes in human neurodegenerative disorders; focus on Alzheimer's disease. Trends in PharmacoL Sci., 80–84.

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Rodriguez-Puertas R, Pascual J, Vilaro T and Pazos A (1997) Autoradiographic distribution of Ml, M2, M3 and M4 muscarinic receptor subtypes in Alzheimer's disease. Synapse, 26: 341–350. Sawada Y, Hiraga S, Francis B, Patlak C, Pettigrew K, Ito K, Owens E, Gibson R, Reba R, Eckelman W, et al. (1990) Kinetic analysis of 3-quinuclidinyl 4-['25I]iodobenzilate transport and specific binding to muscarinic acetylcholine receptor in rat brain in vivo: implications for human studies [see comments]. J. Cereb. Blood Flow &Metab. 10(6): 781–807. Skovronsky DM, Zhang B, Kung MP, Kung HF, Trojanowski JQ and Lee VM (2000) In vivo detection of amyloid plaques in a mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. USA 97(13): 7609-14. St. George-Hyslop P (2000) Molecular genetics of Alzheimer's Disease. Biol. Psychiatary 47: 183-199. Stein W (1986) Transport and diffusion across cell membrane. New York, Academic Press Inc. Vera CR, Woodle ES and Stadalnik RC (1989) Kinetic sensitivity of a receptor-binding radiopharmaceutical: Technetium-99m galactosyl-neoglycoalbumin. J. Nucl. Med., 30, 1519-1530. Weinberger DR, Gibson R, Coppola R, Jones DW, Molchan S, Sunderland T, Berman KF and Reba RC (1991) The distribution of cerebral muscarinic acetylcholine receptors in vivo in patients with dementia. A controlled study with 123IQNB and single photon emission computed tomography [see comments]. Arch Neurol. 48(2): 169-76. Weinberger DR, Jones D, Reba RC, Mann U, Coppola R, Gibson R, Gorey J, Braun A and Chase TN (1992) A comparison of FDG PET and IQNB SPECT in normal subjects and in patients with dementia. J. Neuropsychiatry Clin. Neurosci. ,4(3), 239-248. Yamamura HI, Kuhar MJ, Greenberg D and Snyder SH (1974) Muscarinic cholinergic receptor binding: regional distribution in monkey brain. Brain Research,(66) 541-546.

17. STRATEGIES FOR QUANTIFYING PET IMAGING DATA FROM TRACER STUDIES OF BRAIN RECEPTORS AND ENZYMES JEAN LOGAN Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

INTRODUCTION Positron emission tomography (PET) and single photon emission computed tomography (SPECT) have made possible the in vivo investigation of neuroreceptors, transporters and enzymes that are implicated in disease states such as schizophrenia, Parkinson's disease as well as in addiction and processes associated with aging. Some of the tracers used and the receptors/transporters that they label are: [HC]raclopride (Farde et al., 1989;Volkow et al., 1993; Hietala et al., 1999), [HC]N-methylspiperone (Wong et al, 1986a,1986b), [123I]epidepride (Fujita et al, 1999) for the D2 dopamine receptor; [nC]-Schering 23390 (Farde et al,, 1987; Suhara et al 1991), [ U C]NNC 112 (Abi-Dargham et al., 2000), for the Dl receptor; [HC]benztropine (Dewey et al, 1993a), scopolamine (Frey et al., 1992), [ U C] tropanyl benzilate (Koeppe et al., 1994), [ U C] NMPB (Zubieta et al., 1998), [I8F]FP-TZTP, (Carson et al, 1998) for the muscarinic cholinergic system; [UC] cocaine (Fowler et al, 1989) [uC]d-threo methlyphenidate (Volkow et al., 1995; Ding et al., 1994, 1997), for the dopamine transporter; [HC]dihydrotetrabenazine (Koeppe et al., 1995), for the vesicular monoamine transporter; [1!C]carfentanil (Frost et al., 1989) for the opiate receptor; [UC]WAY-100635 (Mathis et a/,.1994; Farde et al., 1998), for the 5HT1A; [nC]flumazenil (Price et al, 1993) and [123I] iomazenil (Bremner et al, 2000) for the benzodiazepine receptor. Examples of ligands used for the study of brain enzymes are [HC]L-deprenyl and [HC]L-deprenyl-D2 (MAO B) (Fowler et al, 1987, 1995), ["Clclorgyline (MAO A) (Fowler et al, 1987), and [UC] PMP (acetylcholinesterase) (Koeppe et al, 1999). In order to allow comparisons between subjects of measures related to receptor concentration, it is necessary to separate physiological process related to receptor concentration from other processes that influence tracer uptake. In order to do this many methods of varying complexity have been developed and applied. Many are based on one or two tissue compartment models in which uptake into tissue is driven by the plasma concentration of the labelled tracer.

The measurements are radioactivity concentration in tissue

(PET/SPECT) and plasma radioactivity which is divided into that due to unchanged tracer and its metabolites. In some cases these have been simplified so that a reference region (a region of interest (ROI) from the PET study that is devoid of the receptor/transporter being studied) is used in place of an input function. Also some techniques don't require full dynamic scanning relying on an equilibrium between tissue and blood.

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These models are very simple and most likely represent a combination of processes particularly in the case of enzyme inactivation as occurs with ["CJdeprenyl and MAO B which is actually a multistep process. Due to the fact that only the total tissue radioactivity can be measured and not its components, the number of identifiable model parameters is limited- generally to no more than 4, if that. While there are techniques for separately evaluating receptor number and affinity, these experiments are difficult and require the administration of a sufficient amount of drug to block a substantial fraction of receptors. While these studies have the potential for providing important information they will not be discussed here. Most PET/SPECT experiments, particularly in a clinical setting, are in the high specific activity range and the model parameters for reversibly binding ligands are some measure of Bmax'/Kd where Bmax' is taken to be the free receptor concentration and Kd is the receptor-ligand equilibrium dissociation constant. When the free receptor/enzyme concentration does not change over the course of the experiment, the model equations are first order linear differential equations which can be solved either numerically or in closed form. Even if the receptor concentration is changing due to a change in neurotransmitter concentration from a drug treatment, these equations can still be solved in the same way but the model parameter related to Bmax' represents some average over the course of the experiment There are a number of approaches that have been used to evaluate the model parameters. These range from the most complex - optimizing model parameters by solving the model differential equations, a nonlinear least squares approach (NLLSQ), to a ratio of tissue activity in an equilibrium measurement. There are a number of modifications that have been applied to the set of differential equations that simplify the modeling process and in some cases eliminate the necessity of measuring an arterial input function.

A review of various modeling techniques and their

strengths and weaknesses is the subject of this chapter.

MODELS General models for the description of tracer distribution and binding are given below. Cp represents the plasma concentration of labelled tracer, CF is the free concentration of tracer in brain tissue, CNS is the nonspecifically bound tracer. KI and k2 are the ligand transport constants, plasma to tissue and tissue to plasma, respectively. Model la represents regions without specific binding sites. Model Ha adds specifically bound tracer, C$. Here it is assumed that there is only one kind of receptor binding site.

STRATEGIES FOR QUANTIFYING PET IMAGING DATA Blood

Brain

503

^NS

Model la

Model Ha

The binding parameters kNS'and kNS" in Models la and Ha represent nonspecific (nonsaturable) binding, k3 represents (saturable) binding to specific receptor/transporters and k4 is the receptor-ligand dissociation constant. A simplification of models I and II is that the constants describing nonspecific binding (kNS'and kNS") are sufficiently greater than the other kinetic constants that the concentration of free ligand is a constant fraction of the total (free plus nonspecifically bound), that is Cp-fNs C, where fNS is the free fraction (Mintun et al., 1984). With this assumption the models become

Model Ib

Model lib

However, it has been found in a number of cases that it is necessary to use 2 tissue compartments to described the cerebellum (reference region) (for example Abi-Dargham et al., 2000; Logan et al, 1991; Carson et al., 1998 ) so that the nonspecific consists of two parts - one rapid and one slower with binding constants k5 and k6.

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Model Ic

There is the question of whether or not this additional binding is also present in the receptor containing region. In fact, Seeman et al. (1990) reported that nonspecific binding of raclopride is greater in the basal ganglia than in cerebellum.

In some cases the binding is irreversible on the time scale of the experiment so that the model becomes

Model IIIb

The transport constants KI (plasma to tissue) and k2 (tissue to plasma) are functions of blood flow and the permeability surface area product, PS. Based on a homogeneous single capillary model in which tissue concentration surrounding the capillary is constant over the time of capillary transit, the transport constants can be related to F and PS as (Crone, 1963; Renkin, 1959; Kety 1951; Patlak & Fenstermacher, 1975)

PS,

(1)

More complex models relating tracer transport and flow have been considered by Sawada et al., (1991). The distinction between PS] and PS2 involves the incorporation of the free fraction in plasma and tissue, that is PS]=PSfp where fp is the free fraction of ligand in plasma and PS2=*PSfNS with fNS being the free fraction in tissue. The use of fp and fNS presumes that equilibrium between free and bound ligand is rapidly achieved on a time scale shorter than the capillary transit time. The unbound ligand is assumed to pass through the blood brain-barrier by passive diffusion.

The differential equations of Model lib, the commonly used model for receptor binding, are given by

^— = K,Cp(t) - k2C?s - konfNS (B max- L - NB ] dt (2)

STRATEGIES FOR QUANTIFYING PET IMAGING DATA J/^S

dt The assumption implicit in the model of specific binding is that the receptor occupancy is unchanged during the course of the experiment. The parameter k3 is given by k3=/NSA:011(Bmax-NB-L) where Bmax is the total receptor/transporter concentration and NB is the endogenous neurotransmitter concentration.

L is the

concentration of unlabelled ligand bound to receptors. In the high specific activity limit L is negligible compared to Bmax and is neglected. On the other hand NB is generally not negligible and can influence the amount of tracer that binds to receptors and hence the measure of receptor availability. How NB affects the number of binding sites available to the tracer ligand depends upon several factors. If NB and the labelled tracer (L*) both bind to the same site or to overlapping sites on the receptor, then L* will reflect the reduced number of sites. If they don't bind to the same site, then L* will reflect more the total number of sites, although if it doesn't bind to the functional site, it may not necessarily be a good measure of a functional receptor. In many PET studies drug induced neurotransmitter changes are monitored with tracer ligands. Neurotransmitter competition with labelled tracers has been studied by Dewey et al, (1990, 1993a, 1993b), Innis et al., (1992), Volkow et al., (1994), Laruelle et al., (1997) and others. In these cases NB is changing with time (Ne(t)). Although the model equations are no longer linear, they can be solved as though they were by assuming a "constant" Bmax'=Bmax-NB. Endres and Carson (1998) have considered through simulations how binding characteristics of the tracer affect sensitivity to the changes in neurotransmitter concentration. These models are certainly simplifications of the actual binding processes. For example, the binding of [! !C]L-deprenyl which is an irreversible inhibitor of the enzyme MAO B, is thought to react with the enzyme in the multistep process shown below

Cp

~w k'2

w

S+E

SE

k'4

'inact

However, since PET measures the sum of all radioactivity sources within an ROI, it is not possible to uniquely determine kinetic constants for all the steps in the process. As a result, the simpler irreversible model with one binding parameter is used for the description of the binding of [ !1 C]-L deprenyl. When the concentration E-S is approximately constant, then fc3 of the irreversible two tissue compartment model (Illb) corresponds to k'skV^+k's) in the above scheme.

506

HANDBOOK OF RADIOPHARMACEUTICALS

Also some ligands are known to bind to more than one receptor/binding site, for example, N-methyl spiperone (NMSP) binds to serotonin receptors as well as dopamine D2 receptors. complicating factor is the presence of multiple receptor affinity states.

Another possible

Additional binding sites could in

u

theory be added to the model but as with [ C]L-deprenyl without additional information it would be difficult to separately identify all components. In such cases k3 and k4 would reflect multiple components of specific binding. In general the models used to describe PET data represent a macroscopic average of many microscopic processes. The ligand diffuses to the binding site, binds to a receptor, dissociates and rebinds perhaps before diffusing away. Among other things the configuration of the binding sites can contribute to the macroscopic model parameter. There are also some experimental results that suggest that for some ligands the classical occupancy model based on the free receptor concentration given by Bmax - NB is not correct. For example, a decrease in binding of the D2 ligand NMSP after reserpine treatment which decreases synaptic DA was observed (Inoue et al, 1991). Furthermore [3H]NMSP binding was increased after MK801 treatment but [3H]raclopride binding was not significantly changed (Inoue et al., 1999a). Inoue et al., (1999b) offer evidence that these ligands bind to different sites on the receptor. Furthermore the receptors appear to form dimers and larger clusters. Differences in the binding properties may be due to the different binding capacities to D2 receptor dimers and monomers. Zawarynski et al., (1998) found that a spiperone derivative labelled only the D2 monomer and a raclopride derivative both the dimer and the monomer. These results and others relating to the occupancy model are discussed by Laruelle (2000). In any case, for many ligands the models described above provide a useful tool for comparing data although they are certainly an approximation to the underlying physiological processes. STRATEGIES FOR DETERMINING MODEL PARAMETERS MEASURES OF RECEPTOR AVAILABILITY Rather than compare individual model parameters which are subject to considerable variability (Carson et al., 1993), comparison among subjects is usually made by comparing a composite parameter that is a combination of model parameters. For reversible ligands this is either the binding potential (BP) (Mintun et al., 1984), the total tissue distribution volume (DV), the distribution volume ratio (DVR) (the ratio of the DV of a receptor region to that of a reference region without the receptor) or an effective binding potential derived from the DVR. Another possibility is the difference between the receptor DV (DVRO|) and the DV of the reference region (DVREF). All of these measures are a function of the free receptor binding sites but they each depend upon assumptions about the constancy of other processes. The binding potential is defined as BP =Bmax'/A^ (Mintun et al, 1984). In terms of the model parameters used here it is given by k^/(k^s) since &3 implicitly contains the free fraction (f NS ) (&3=£onBmax'/Ns and Kd^k^k^). (In Mintun et al., 1984,

STRATEGIES FOR QUANTIFYING PET IMAGING DATA

507

/NS=/2.) Many researchers define the binding potential as BP^/k^ =Bmax' IKd' where Kd' includes /NS (Kd'= k0ff/(k0n /NS) **Kd/fus)-

1° this case, the assumption is that /NS

is constant and does not contribute to

differences in BP. The distribution volume is given by the ratio of the tissue to plasma under equilibrium conditions, that is DV=CROi/Cp, CRQI is the tissue concentration of a region of interest. For some ligands equilibrium can be achieved and the DV can be measured in this manner (see below). Alternatively, the DV given by (Lassen & Perl, 1979) as

]cROl(t)dt

is valid for non -equilibrium experimental conditions. In most cases this is not a practical approach to calculating the DV. The DV can be determined under non-equilibrium conditions directly by graphical analysis described below. The DV can also be determined from the model parameters which for the 1 and 2 tissue compartment models (Model Ib and Model (lib)) are Kj/k 2 and K]/k 2 (l +k3/k4), respectively. If there is a component of slow nonspecific binding in both, then these become Ki/k2(l4-k3/k4+k5/k6) and K1/k2(l+ks/k6). Since the transport constants are a function of plasma protein binding (PSi=PS/p in Eq(l)), the DV also depends upon plasma protein binding. This can be eliminated by independently measuring fy and removing it from the DV (DV/ fp) (Carson et al., 1993). The problem is that generally a large fraction of ligand is bound to plasma proteins so that errors in the determination of Jp can introduce considerable variability into the DV. By basing comparisons on the distribution volume ratio (DVR) the dependence upon plasma protein binding is removed.

The DV's can be determined directly by graphical analysis or

equilibrium measurement for example. The DVR is then (assuming that the ratio of transport constants is the same for both receptor and reference region)

DVR =

DVREF

k,

Kd'

The BP can then be calculated indirectly as DVR-1 as opposed to explicitly determining k3 and k4. The DVR is still a function of nonspecific binding through /Ns in Kd'. If there is a component of slow nonspecific binding in both regions, then the DVR expressed in terms of model parameters is

508

HANDBOOK OF RADIOPHARMACEUTICALS =

K

REF

» *S

(Kl/k2)k3/k4

where k5 and J(% refer to the "slow" component of nonspecific binding (see Model Ic). If the ratios K\/k2 and ks/kt are the same for both regions, then the binding potential derived from the DVR is BP" where f

= 1/(1 + k5

/ k6

DVR-l^f'k^/k4

) and /?N$ is 1 . Therefore the BP calculated directly by estimating the

model parameters could lead to a different value from BP calculated indirectly through the DVR. If a slow component exists in the receptor compartment also, it would most likely be difficult to separate from the receptor binding component and the BP calculated using Model lib would include both components, overestimating the BP. Subtracting the reference DV from the ROI DV gives K{/k2(BP) defining BP as £3/Jt4. This measure is dependent upon fp through K1 but the dependence upon fNS has been removed since it appears in both k2 and k3 and therefore cancels (see Carson et al., 1997). A comparison of outcome measures for equilibrium, kinetic and graphical methods is given by Laruelle (2000) (see Table 6 in that article). For irreversibly binding ligands, receptor availability is contained in model parameter k3. Logan et al., (2000a) found that reproducibility on test/retest for [HC]L-deprenyl-D2 significantly improved if comparison is based on the combination parameter k3 where A=K1/k2. Graphical analysis (Patlak et al., 1983) of uptake data from irreversible ligands provides an influx constant Ki given by K\k^/(k2+ &3) which depends upon blood flow (see below).

MODELING OPTIONS FOR REVERSIBLE LIGANDS WITH A MEASURED PLASMA INPUT FUNCTION The model parameters can be optimized by solving the differential equations using a measured plasma input function and determining the set of values that give the best fit to the data. For a discussion of optimization methods see Carson (1986). The BP and DV can then be determined from the appropriate combination of model parameters. The linearized version of the standard compartment models (Blomqvist, 1984; Evans, 1987) provide a more efficient method of parameter estimation. The model equations become a set of linear equations. For the one and two tissue compartment models the linear equations (for scan times t, ) are

STRATEGIES FOR QUANTIFYING PET IMAGING DATA

509

CROI (ti) = K\ \Cpdt - k2 \CROI (t}dt 0 ', /'

0

',

',• /'

',

^

r

?-K]CROI (t}dt - k2k^CRO, (t}dtdt + K{ jCpdl 00

0

00

0

where K=(k2+k3+k4). A more general approach to linear least squares analysis is given by Thie et al. (1997). This approach is model independent in that the constants can be determined without reference to a particular model. There are 2n coefficients c, in which fits to data can be made with n=l ,2 or 3 and the optimum number selected based on a statistical analyses (Thie et al., 1997). r

T

Tt

Tt

CKOl (T) = c^Cpdt - c2\CROl (t)dt + c3 J \Cpdfdt - c4 J ICKOI (t)didt'+ (if needed) J J J 0

0

00

00

The problem is that the coefficients are function of both blood flow and the receptor parameter. Parameter estimates based on the linear forms of the model equations as in Eq (5) are subject to bias because the equation errors, are not statistically independent that is each succeeding one depends upon the previous one (Feng et al, 1993, 1996).

In order to overcome the bias problem, Feng et al., (1993) introduced a

generalized linear least squares (GLLS) method which removes the bias. The GLLS form of the one tissue model is

Feng et al. have generalized this to more complex models (Feng et al., 1996). In graphical analysis the set of linear equations describing a general model is transformed into a single equation which becomes linear for time t* (Logan et al,

1990).

While this is applicable to a

multicompartment system, only two parameters are determined, the slope and the intercept which are combinations of the model parameters. The graphical analysis equation for points determined by scan times t\

\CROl(t)dt \Cp(t)dt ^ =[DV +Vp]^ + int CROI w ) CROI w )

(7)

510

HANDBOOK OF RADIOPHARMACEUTICALS

where DV+Vp is the slope for the linear region which occurs for times tt >t* and Vp is the contribution of the tissue blood volume. The condition for linearity of Eq(7) is that the intercept (int) which for a two tissue compartment model is given by

1

1+

is constant. For some time f > t ', the compartment concentrations follow the plasma concentration so that (Ci+C2) ec Cp and €2°* Cp (the steady state condition) which insures that int is constant since Cp cancels. In many cases the intercept becomes constant even before, (Ci+C2)/Cp becomes constant.

Therefore the

graphical method can be applied before the steady state condition becomes valid, when for some time t* < t', the ratio C2/(C,+C2) varies slowly and is effectively constant. The limiting value of the time dependent portion of the intercept is given by

C2(/) --

>-

1

. The graphical analysis is illustrated in

Figure 1 using simulated data with the same DV but with very different kinetics. For the upper curve (Figure la), the main contribution to the DV is from the ratio of transport constants 'k=K1/k2 while for the lower curve the main contribution is from the ratio of binding constants (k3/k4 =20 and 5). The graphical analysis is illustrated in Figure Ib. Both achieve linearity but with very different times t* which will affect the length of scanning time required to obtain an accurate estimate of the DV. The graphical method has been extended by Ichise et al., (1999) to account for labelled lipophilic metabolites which could cross the blood-brain barrier, interfering with the quantification of ligand uptake. Alternatively the DV can be obtained directly by manipulating the plasma levels so that equilibrium is reached.

Patlak and Petigrew (1976) developed a method for obtaining infusion schedules to achieve

specified blood concentration levels over time. This method has been used in particular to produce a constant input function. Carson et al., (1993) extended this method to include a bolus injection with a continuous infusion to produce a true equilibrium so that the true DV is given by the ratio of tissue to plasma. The advantage is that only a few scans are required and arterial blood sampling is not necessary. Whether this method is appropriate depends upon the kinetics of the tracer. (CT)

A transient equilibrium between tissue

and plasma can be achieved after a bolus injection so that Cr(t)/Cp(t) is constant. This however is

generally not the true distribution volume but is a function of the rate of plasma clearance (Carson et al., 1993; Logan et al., 1990).

STRATEGIES FOR QUANTIFYING PET IMAGING DATA

511

WITHOUT A MEASURED PLASMA INPUT FUNCTION There are several approaches to determining model parameters without an input function. These methods require a reference region, a region devoid of the receptor/transporter or other binding site being studied. Lammeitsma et al., (1996) presented a reference region method assuming the reference region could be described by a one tissue compartment model.

They derived the following relationship between

concentration of tracer in the reference region, CKEF and Cr, the total tissue concentration for a two compartment model

CT (0 = R, [CREF (0 + aCREF (r) <8> exp(-cf) + bCREF (t) <8> exp(-dr)]

(8)

where R\ is the ratio K1 / K1REF , and a,b,c,and d are combinations of the model parameters, k2, k3, and k4 and are determined by standard nonlinear regression analysis. A simplified reference tissue model which assumes that the receptor region can also be described by a one tissue compartment is given by the equation (Lammertsma & Hume, 1996)

Cr(0 = R,CREF (t} + [k2 -R}k21(1 + BP)]:REF (r) <8>exp(-*2r/(I + BP^

<9>

in which three parameters, k2, RI and BP, are to be determined using nonlinear analysis. Gunn et al., (1997) revised this method so that two parameters R) and P are determined using a linear least squares optimization for a set of values of y

CT (r) = R,CREF (0 + pCREF (0 <8> expHO j and p are composite parameters corresponding to Eq(9). An alternative linearization of the simplified reference tissue method was given by Logan et al., (2001 a) as C5(0-*V*V ®C](t) = -k2e-i>'Cl(t) + ^CREF(t)~-^(k2 A,

-k«EF}e-k* ®CREF(t) (lO)

A,

This method is based on the generalized linear least squares method of Feng et al., (1993). There are three constants to be determined by a linear solution, k2 K} / Kl

F

, and (K{ I KfEF)(k2

initial estimate, k2 . Generally only a few iterations are required (Feng et al., 1993).

—k2F} given an

512

HANDBOOK OF RADIOPHARMACEUTICALS

Another method based on a reference region input is a modification of the graphical analysis method (Logan et al., 1996). The DVR can be calculated directly with the graphical method by using data from a reference region (CREp (tj) with an average tissue to plasma efflux constant, ki to approximate the plasma integral

]cRO](t)dt -2

J C- REF

(*)

= DVR

REF

+ int'

(ii)

where int' is int + 5, 5 is the error term given by

When

DVR CREF(T}

.

is small and/or reasonably constant the term containing k*EF in Eq(l 1) can be

neglected.

Ichise has proposed an alternative to Eq(l1) which is a multilinear regression (Ichise et al., 1996). This method appears to provide the same results as Eq(l1) with k2 =

. When lipophilic metabolites are present

in tissue, Ichise has proposed a method requiring a single blood sample to generate the DV from the DVR calculated using the reference tissue with the contaminating metabolites (Ichise, et al., 1999). Farde et al., (1986, 1989) determined Bmax'/Kd' for raclopride at a transient equilibrium point of the specifically bound ligand (when dCs2 /dt = 0 ). Cs2 was defined as the difference between the radioactivity in the putamen and that in the cerebellum. At high specific activity Bmax/Kd' = C2 /Cl

where the free ligand (C,

) is the cerebellar

(reference region) concentration at the point dC2 /dt = 0 . Farde et al., (1989) also used this technique to estimate separately Bmax' and Kd' by doing additional studies at lower specific activities and using a Scatchard analysis. Although this method is vulnerable to errors in the determination of the point at which dC2 /dt = 0 , comparable values of Bmax' and Kd' were found for both the pseudo equilibrium and kinetic methods for [' 'C]raclopride. Another factor limiting the usefulness of this technique to other tracers is error in the estimation of C2 due to a difference in blood flow between the two regions (Logan et al., 1997).

STRATEGIES FOR QUANTIFYING PET IMAGING DATA

513

MODELING OPTIONS FOR IRREVERSIBLE LIGANDS

WITH A MEASURED PLASMA INPUT FUNCTION Irreversibly binding ligands (Model Illb) are essentially trapped for the time course of the scanning procedure. Information about receptor availability is contained in model parameter &3. The three model parameters can be estimated using an optimization procedure and solving the differential equations directly. Different approaches to optimizing £3 are illustrated in Koeppe et al., (1999) for the ligand [I!C]PMP which binds irreversibly to acetylcholinesterase. These included unconstrained estimation of all three parameters and constrained estimation of k^ by fixing the K\lki ratio. This assumes that the ratio is relatively constant across the brain, an assumption which has been found to hold for a number of PET tracers.

Alternatively, a model independent graphical method (Blasberg et al., 1979; Gjedde 1981; Patlak et al. ,1983; Patlak & Blasberg, 1985) evaluates the rate constant (Ki) for the transfer of tracer from plasma to the irreversible compartment. The equation for this is

Cp(i)dt Cp(T) which is linear for the times T>t* when Ve, the distribution volume of the reversible part (the ratio of the concentration in the reversible compartment to plasma) is constant (for Model Illb this is C 1 /Cp). Relating this to the two tissue compartment irreversible model, the influx constant Ki can be expressed as Ki is expressed in terms of two parameters, K1 which represents the transport of ligand from plasma to tissue and the combination parameter Ak3 which also contains the ratio of transport constants (k*=K\!ki). Although KI and k2 are functions of blood flow, X is not. From Eq(5) it can be seen that Ki depends upon K, (blood flow) as well as free receptor/enzyme concentration (contained in Ak3), Only if k2 »

kj so that Ki —>Akj, is Ki independent of blood flow. Therefore in order to extract a parameter

independent of blood flow (ligand transport) it is necessary to determine K1. Ak3 can then be determined from Eq(13)

Kt Ki

514

HANDBOOK OF RADIOPHARMACEUTICALS

Wong et al., (1986a) have used a variation of the graphical analysis for the estimation of model parameters for the dopamine D2 ligand [MC]NMSP which appears to bind irreversibly over the time period of the experiment. In this modification the early part of the curve (before the linear phase) is used also to estimate parameters. The analysis equation uses the normalized time integral of plasma radioactivities, 0 given by T

&=I o and the tissue plasma ratio (V(T))

Cp(T)

3

where p = ks/ks accounts for a reversible component of either low specific or nonspecific binding in the receptor region. In the case of NMSP there are reference regions such as the cerebellum without specific K?EF binding from which A=—^77- is determined (when V(T)=CKEF{T)/Cp(T)).

The transition of V(T) vs 6(T) to

a linear phase at later times is determined by T. When X is known the model has three parameters, k2, k3 and p. It is assumed that p is not present in the reference region.

WITHOUT PLASMA INPUT When the concentration of original tracer in plasma reaches zero during the time of the study, k$ may be estimated entirely from the shape of the time activity curve (TAG) (Frey et al., 1997a,b). This method was applied to estimation of acteylcholinesterase activity and was found to be suitable for regions of low enzyme activity (Koeppe et al., 1999).

Patlak and Blasberg (1985) extended the graphical analysis for irreversible ligands to an analysis using a reference region in place of the plasma input It is assumed that the reference region has no specific binding so that in the steady state condition CREF (t>t*.

i,

\CREF(t)dt ROl

, When this is true so that a plot of CR0^t^lCKEF{t^ vs J CREF (f)dt CREF (t, ) is a straight line for t,.> t* with slope o / K' = Ki/(DVREF +Vp') where Vp' is the blood volume of the reference region and DV^ reference region.

is the DV of the

STRATEGIES FOR QUANTIFYING PET IMAGING DATA

515

SENSITIVITY OF THE OUTCOME MEASURE The outcome measure must be sufficiently sensitive to variations in the underlying receptor availability to accurately register changes. This translates into particular requirements of tracer ligand kinetics. In the case of reversibly binding ligands, the binding potential (k 3 /k 4 ) needs to be sufficiently greater than one so that it can be reliably estimated. If it is too small there will be little difference between the reference region with no receptor concentration DV=K 1 / k2 and the region with a receptor density DV= K 1 /k 2 (l+k 3 /k 4 ). On the other hand if BP is too large, it may become difficult to obtain an estimate of the DV in the time span of the experiment. In particular, if k3 >> k2 either due to a high affinity (kon) or a large Bmax or slow tissue to plasma efflux, then the concentration of ligand bound to receptor is limited by tracer delivery, a situation referred to as "flow limited". This leads to an underestimation of the DV (or a large uncertainty in the DV) or if using an irreversible model, the receptor parameter k3 is contaminated by delivery effects (see discussion in Koeppe et al., 1994). This has been a problem for some of the muscarinic cholinergic ligands such as scopolamine (Frey et al., 1992) and less of a problem although still present for [HC]tropanyl benzilate (Koeppe et al., 1994) and [' 'Qbenztropine (Dewey et al., 1993a). Figure 2 illustrates the sensitivity of the TAC of a simulated irreversible ligand to variations in the receptor binding parameter, k$. Each curve from &3=.0033 to 0.65 increases k3 by 50% over the previous one (K 1 =A5 mL/min/mL, k2-.G75 min '). The bottom curve has no receptor binding (k3=0). For very small values of k3. there is little change in the TAC with a relatively large change in k3. The presence of even a small amount of noise would make it difficult to distinguish differences at this level. The maximum change occurs in the middle region. At the high end, when k3 » k2 there is little change in the TAC with a large change in k3 and the binding is "flow limited". (The flow limited condition can also be expressed in terms of Ki, (see Eql 3)). When KI ~ Ki, only one parameter can be determined, KI , and no information can be obtained about enzyme/receptor concentration.). In the regions of higher specific binding the estimates of k3 become much more variable, although they are highly correlated to k2 (Logan et al., 2000a; Koeppe et al., 1999). In order to reduce this variability. Fowler et al., (1995) have used the parameter /k3 which is much more stable since it contains the ratio k^ik^. Alternatively Koeppe et al., (1999) proposed using a scheme in which all three parameters are determined but the ratio Ak$ is scaled to the value of A determined from a region of low specific binding thus giving a scaled value of &3.

516

HANDBOOK OF RADIOPHARMACEUTICALS

The sensitivity of irreversible ligands that are close to the flow limit can be improved by reducing the binding parameter, £3. This was done with the tracer [nC]L-deprenyl by substituting deuterium for hydrogen at the reaction site. This is an example of the kinetic isotope effect in which the increased mass of the atom involved in the reaction slows the reaction rate. Figure 3 illustrates two uptake curves from [uC]L-deprenyl H2 and ["C]L-deprenyl-D2 in the same subject. The difference between K, and Ki is significantly greater for the D2 compound than for the H2 compound, 0.3 and 0.12 (mL min ' mL') respectively. In other regions of interest with higher MAO B concentration the H2 difference was found to be even smaller. The sensitivity of H2 to differences in MAO B concentration is much less than for the D2 ligand. This leads to greater variability in model parameters. WHICH MODEL? The model structure that best describes a data set is not necessarily driven by the presence of multiple types of binding. For example, a ROI from a receptor containing region could be described by a one tissue compartment model even though there is specific binding to the receptor as well as nonspecific binding. Why the binding kinetics of one ligand requires a two-compartment model while the kinetics of another does not has to do with the impulse response function of the two-compartment model given by

A, =

K]

—(k,+k4-al}

a, -a,

A2 =

K

!— (a 2 -* 3 -* 4 )

a, -a.

A, exp(—CC\i) + A2 exp(—a2t) (Carson et al., 1998) where A|.2 and ct|,2 are combinations of Kifafaand &4. If one of the exponential terms dominate, then a one compartment model will adequately describe the data. Following (Carson et al., 1998) whether a two compartment fit is required can be determined by considering the fraction of the area of the response function due to the second term for time T, that is

A 2 (l-exp(-tt 2 r))/g 2 A, (1 - exp(-a,r)) / a{ + A2 (1 - exp(-«2r)) / a2 The effect of varying the binding parameters Jt3 and fc4 (while maintaining a constant binding potential, kj/k4) on the integrated response function ratio of Eq 14 is shown in Figure 4. Using model parameters similar to those found for [' 'CJraclopride with Af|=.15 and Jt2=.36 for all simulations giving DV = 1.917 (DV, indicates the DV generated by fitting the data to a one tissue compartment model with two parameters, DV2 a two tissue model with four parameters). The maximum time was 60 min. DV| underestimates the true DV for the lower values of Jt3 and Jt4. This is improved somewhat by extending the analysis time to 95 min for which DV, becomes 1.59 for £3=0.09. The graphical DV's for the 4 cases were 1.80, 1.89, 1.92, 1.92. Extending the time to 95 min the DVG was found to be 1.89 and 1.91 for fc3=0.09 and 0.18 min ' respectively. There

STRATEGIES FOR QUANTIFYING PET IMAGING DATA

517

was no change in the other two. For the simulations with fc3=0.09 and 0.18 a two compartment model is required to recover the true DV using the NLLSQ method. For the two simulations with higher values of £3 and &4, DVi is close to the true DV and the addition of another tissue compartment would not be justified since the parameters would most likely not be identifiable in the presence of noise. There are also instances in which the "nonspecific" reference regions are better described by a two tissue compartment model. This has been observed for some studies with the radioligands ["CJraclopride (for example Logan, 2001 b), and [I8F]spiperone (Logan et al., 1987). Also Abi-Dargham et al., (2000) observed that a two compartment model gave a somewhat better fit to cerebellar data for the DI ligand [ U C]NNC 112. From 16 studies in the baboon with [l!C]raclopride the DVi underestimated the DV compared to DV 2 and to DVG, DV,/DV2=.83±.05 and DVo/DV2=.98±.02 (Logan et al., 2001 b). This appears not to be related to specific binding. This apparent additional binding could be due to an error in the metabolite correction of the input function. If the fraction of original tracer is small at later times, then a small error in the metabolite correction will result in a large difference in the plasma concentration (Carson et al., 1998). Also uptake of a small quantity of lipophilic metabolites at later times will result in a bias in the model. Whether the second compartment in these nonspecific regions is due to this or is in fact a true slow nonspecific binding is unclear. Also another issue is whether it is also present in the ROI under study and should be taken into account, REFERENCE TISSUE METHODS Sossi et al., (2000) compared BP estimates from the graphical tissue input method and the simplified reference tissue method (SRTM) for 4 ligands, [IlC]methylphenidate (DA transporter),[uC] dihydrotetrabenazine (DA vesicular transporter), [' !C] raclopride (D2 antagonist) and ["ClSchering 23390 (Dl antagonist), finding nearly identical results for both methods and similar reliability and reproducibility. The BP estimates were somewhat lower than those from compartment analysis. BP from compartmental analysis were derived indirectly from the DV's for the receptor and reference region so that BP=(DVRorDVREF)/DVREF.

The model used was the one tissue compartment model. Both the

reference and receptor regions for these ligands could be described adequately by a one tissue compartment model satisfying the basic assumption of the simplified reference tissue method (SRTM).

Slifstein et al., (2000) compared the SRTM BP's with those from standard compartmental modeling using simulations with arterial input functions for the 5HT1A tracer [UC]WAY100635 and [ H C]NNC112, a Dl receptor tracer. When the reference region was a single compartment the SRTM overestimated BP by 5 to 15%,

However, the assumption in the SRTM is that the receptor region can also be described with a single

tissue compartment. The overestimate may be related to this. An additional compartment in the reference region also distorted the results as one would expect with the SRTM underestimating the BP, also differences

518

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in flow between the two regions affected the BP. There is the additional complication that the receptor region may also contain the slow nonspecific binding. Alpert et al., (2000) also tested the SRTM with simulations using a two tissue compartment model and a measured input function with labeled altropane, a dopamine transporter ligand. The reference region was generated with a single tissue compartment model. For the receptor ROI tf^.38 k2=.l 5 and BP was fixed but values of kj, and fc4 were allowed to vary. For lower values of £3 < .5 min-1 there was deviation from the true BP which became larger as k3 decreased. A likely explanation for this behavior is that for the larger values of &3 and Jt4, a single compartment would describe the data but as *3 deceases, a two compartment model is required which violates the basic assumption of the SRTM. In order to address one of the limitations of the SRTM, Watabe et al., (2000) have proposed a two tissue compartment model to use for the reference region while retaining the one tissue compartment for the ROI. An alternative reference tissue model is used by Acton et al., (1999) for describing ["TcJTRODAT-l binding to DA transporters in baboons using SPECT. The assumptions made were that the transport constants were the same in both the ROI and reference region and that the specific binding component could be extracted by subtracting the reference region from the ROI as in Farde's pseudoequilibrium method. The constraint of having the same transport parameters for both regions is not likely to be valid for all ligands limiting the usefulness of this technique. The reference region method was lower than the BP derived using the compartment model but the same constraint was used in the model.

Also it is unlikely that the

specifically bound is accurately represented by the difference between the ROI and reference region over the time course of the study (Logan, et al., 1997). BIAS IN THE GRAPHICAL ANALYSIS METHOD The graphical analysis method is a useful tool for rapidly obtaining information about the binding of radioligands. The strength of the method is that it does not require a particular model structure. However, since it is derived from the linearized compartmental equations, it also displays a bias in the case of noisy data resulting in the underestimation of the slope (DV) and the underestimation is greater with larger DV's (Hsu et al., 1997; Slifstein et al., 1999; Abi-Dargham et al, 2000). In order to remove the bias, Logan et al., (2001 a) have proposed a modification of the GLLS method developed by Feng et al., (1993) to use as a smoothing technique for more general classes of model structures. The one compartment GLLS method was applied to the data in two parts, that is one set of parameters was determined for times 0 to T] and a second set from T| to the end time. The curve generated from these two sets of parameters was then used as input to the graphical method. This was been tested using simulations of data similar to that of the PET ligand [ M C]-

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d-r/ireo-methylphenidate (MP, DV=35. mL/mL) and [HC]raclopride (RAC, DV=4.92 mL/mL) with the result that in the case of moderate noise the bias was substantially removed.

This combination of the GLLS

method and the graphical method provides the possibility of retaining the model independent type of analysis without the bias inherent in the linear methods while still maintaining a fairly simple method of analysis. This method was also applied to the simplified reference tissue model of Lammertsma and Hume (1996). The equation was modified to allow a linear solution for #2 as in Feng's method. Estimates of three parameters were generated in this case as opposed to two when the input function is measured. The same two part procedure was used to smooth the data as was done with the DV and the graphical method was applied to the smoothed data using the reference region and an average efflux constant (Logan et al., 1996). CONSTRUCTION OF PARAMETRIC IMAGES Reliable image wide parameter estimation methods are important because of the potential increased information content of parametric images over ROI analysis, although both are important. One desirable characteristic of image wide parameter estimation methods is that they perform well in the presence of noise which is considerably greater than in ROIs. Other desirable characteristics include speed of the calculation since it must be done for all voxels in the image, and model independence of the method since there will generally be variations in specific binding so that voxels in one structure may require a different model from voxels in another structure. The weighted least squares method (Alpert et al., 1984; Koeppe et al., 1991) works well when a one compartment model is adequate for all regions. From this method parametric images of ligand transport rate and distribution volume can be constructed. It requires a measured input function. Holthoff et al., (1991) have shown that altering blood flow does not alter the DV and thus demonstrated that the DV obtained in this manner is a measure of specific binding and not ligand transport. Graphical analysis with a measured plasma input function (Logan et al., 1990) is model independent but gives a biased estimate in the presence of significant noise particularly for ligands with high DV's. However, Koeppe et al, (1997) found good agreement between images generated using the weighted least squares and graphical methods for (+)-a-[nC]-dihydrotetrabenazine (DTBZ) which binds to the vesicular rnonoamine transporter (DV in the caudate-putamen was ~ 11 to 12 mL/mL). The smoothing strategy discussed previously may prove to be a means of maintaining the model independence. The method of Gunn et al., (1997) is a modification of the simplified reference tissue model adapted to parametric image construction without a measured plasma input function. The method uses precalculated basis functions for a range of values of the nonlinear model parameter and includes parameter bounds. The assumptions are the same as in the original formulation of the method, that both the reference region and binding regions can be described by a one tissue compartment model (Lammertsma & Hume, 1996). The method was found to work well for ["Qraclopride and ["CJCFT. The presence of additional binding in the

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reference region was tested by simulations and found to underestimate the BP as expected. The graphical method with a reference region input (Logan et al., 19% ) is model independent but is subject to bias in the presence of noise. The previously described adaptation of the simplified reference tissue method used as a smoothing technique prior to applying the graphical analysis has been proposed as a possible solution to the bias problem. The nonlinear least squares methods which are based on a particular model structure generally require considerable computation time as well as being subject to local minima. These methods are not generally used for image wide parameter estimation of DV or BP. The simplicity of the bolus plus constant infusion equilibrium method makes it an attractive alternative although different structures may require different infusion schedules to achieve equilibrium. In this case the method could be used for estimation of BP in voxels within a given structure. For irreversible ligands parametric images of the influx constant Ki can easily be constructed (Patlak et al., 1983, 1985). However, since Ki depends upon blood flow comparisons based on this parameter will be subject to differences in blood flow as well as changes in receptor binding. If changes in transport are known to be important, then the transport constant needs to be estimated. Logan et al. (2000b) have proposed a method for estimating KI from initial part of uptake curve and using Ki to estimate Xk3. Turkheimer et al., (2000) has introduced a new approach to generating parameteric images that is based on a wavelet transform of each image in a dynamic sequence. Linear modeling procedures such as the graphical analyses can be done on the wavelet coefficients which represent a spatial object as opposed to a single pixel. This is followed by thresholding and the application of the inverse wavelet transform to recover the parametric image. Among the examples presented were dynamic PET FDG and ["CJraclopride studies with the result that noise was reduced compared to graphical analyses without the wavelet transform while details of brain structures were preserved. SUMMARY A description of some of the methods used in neuroreceptor imaging to distinguish changes in receptor availability has been presented in this chapter. It is necessary to look beyond regional uptake of the tracer since uptake generally is affected by factors other than the number of receptors for which the tracer has affinity.

An exception is the infusion method producing an equilibrium state. The techniques vary in

complexity some requiring arterial blood measurements of unmetabolized tracer and multiple time uptake data. Others require only a few plasma and uptake measurements and those based on a reference region require no plasma measurements. We have outlined some of the limitations of the different methods. Laruelle (1999) has pointed out that test/retest studies to which various methods can be applied are crucial in

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determining the optimal method for a particular study. The choice of method will also depend upon the application. In a clinical setting, methods not involving arterial blood sampling are generally preferred. In the future techniques for externally measuring arterial plasma radioactivity with only a few blood samples for metabolite correction will extend the modeling options of clinical PET. Also since parametric images can provide information beyond that of ROI analysis, improved techniques for generating such images will be important, particularly for ligands requiring more than a one-compartment model. Techniques such as the wavelet transform proposed by Turkheimer et al., (2000) may prove to be important in reducing noise and improving quantitation. ACKNOWLEDGMENTS This research was carried out at Brookhaven National Laboratory under contract DE-AC02-98CH10886 and with the U. S. Department of Energy and supported by its Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of Neurological Diseases and Stroke (NS 15380).

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18. RADIOPHARMACEUTICALS FOR STUDYING THE HEART DAH-REN HWANG8 AND STEVEN R. BERGMANN

b,c

^Department of Psychiatry; bDivision of Cardiology, Department of Medicine; and 'Department of Radiology, Columbia University, New York, NY 10032, USA.

INTRODUCTION Imaging of the heart with radiotracers has proven extremely valuable not only for clinical diagnosis and decision making, but also for evaluation of physiology under both normal and disease states. Quantification of myocardial perfusion during stress is the most widely performed procedure in nuclear cardiology for both the diagnosis of coronary artery disease and also to evaluate the efficacy of interventional functions. Metabolic imaging using tracers such as 2-[18F]fluorodeoxyglucose (FDG) and fatty acids is useful for delineating the metabolic abnormalities that underlie cardiac dysfunction. The alterations that occur with ischemia have been used to delineate viable from nonviable myocardium. Newer agents that delineate myocardial hypoxia may also prove useful for this function. More recently, use of drug and receptor ligands has enabled evaluation of drug binding as well as the role of sympathetic, parasympathetic, and muscarinic innervation and their alteration with a number of disease states. This chapter will review the most widely used radiopharmaceuticals for evaluating the heart and concentrate on newer agents and future developments. PERFUSION TRACERS Alterations in myocardial perfusion underlie the cardiac dysfunction that occurs in coronary artery disease. Thus, evaluation of myocardial perfusion is critical for the diagnosis of coronary artery disease and for evaluating the effects of both medical and interventional strategies. Because of the paramount role of alterations in myocardial perfusion underlying cardiac dysfunction, it is not surprising that tracers to assess blood flow were among the earliest developed. Initially, because of its avid extraction and retention, potassium-43 was developed as a perfusion tracer. Since the 1970s, thallium-201 (Tl-201) (t1/2 = 73 hr) has been used extensively as myocardial perfusion agent and for assessing myocardial viability (Maddahi et al., 1994). The uptake of these tracers occurs predominantly through the Na+/K+ channel. For Tl-201, the rapid myocardial extraction depends on perfusion, whereas the myocardial retention/redistribution reflects myocardial viability. However, the low energy and the low dose allowed due to half-life considerations led to the development of other tracers (Jain, 1999). In the 1980s, lipophilic technetium-99m (tm - 6.03 hr) complexes were demonstrated as good myocardial perfusion agents. To date, several tracers are commercially available and have been widely used. In general, these Tc-99m tracers can be classified as 1) cationic tracers, such as sestamibi (Cardiolite, a methoxyisobutyl Handbook of Radiopharmaceuticals, Edited by M. J. Welch and C. S. Redvanly. © 2003 John Wiley & Sons, Ltd

HANDBOOK OF RADIOPHARMACEUTICALS

530

isonitrile complex) (Sachdev et al., 1990) and tetrofosmin (Myoview, a (l,2-bis[bis(2ethoxyethyl)phosphino]ethane complex) (Kelly et al., 1993); 2) mixed cationic ligands, such as Q12 (TechneCard) (Lenihan et al., 1999); and 3) neutral complexes, such as teboroxime (CardioTec, a CDOMeB complex)(Narra et al., 1989) and nitrido dithiocarbamate complex (Tc-N-NOET)(Ghezzi et al., 1995; Vanzetto et al., 2000). Currently, sestamibi and tetrofosmin are the only two FDA approved tracers. An excellent review on these agents appeared recently (Jain, 1999). The molecular structures of these tracers are shown in Figure 1.

CNR

1

R R

R=

R

R=

R= -OEt

M
-OMe

Tetrofosmin

Sestamibi

Teboroxime

Q12

TcN-NOEt

Figure 1. Molecular structures of Tc-99m perfusion tracers.

In addition to the single-photon emitting radiopharmaceuticals, several positron-emitting tracers are used routinely for myocardial perfusion studies using PET. These PET tracers not only allow for the diagnosis of coronary artery disease with a high degree of sensitivity and specificity, but using mathematical models also allow quantitative assessment of myocardial perfusion (i.e., in ml/g/min). According to the production methods, these PET tracers can be classified as generator produced, such as rubidium-82 (82Rb) (half-life = 76 seconds) (Gould et al., 1986) and copper-62 (62Cu) -PTSM (half-life = 9.74 min) (Green et al., 1990; Herrero et al., 1996), or cyclotron produced, such as Oxygen-15 (15O) water (t )/2 = 2 min) and Nitrogen-13 (13N) ammonia (t, /2 = 10 min). For 82Rb, 62Cu-PTSM, and 13N-ammonia, the relation between blood flow and uptake is nonlinear with decreasing myocardial extraction at higher flow rates. Therefore, these tracers may

RADIOPHARMACEUTICALS FOR STUDYING THE HEART

531

lead to an underestimation of coronary flow if only qualitative imaging is applied. Nonetheless, these tracers provide good-to-excellent images of the heart and have been used extensively. l3N-ammonia is readily produced via a cyclotron by bombarding a water target (containing 10 mM of ethanol) with proton beam (Suzuki &Yoshida, 1999). 13N-ammonia provides excellent quality PET images, and its 10-min half-life is very convenient for clinical studies (Schwaiger & Muzik, 1991). 15O-water diffuses freely across the myocyte and the uptake is not dependent on metabolic trapping. It is thus an ideal flow tracer from this point of view and has been used for quantifying myocardial blood flow in humans (Bergmann, 1997). 15O-water is readily produced by passing 15O-oxygen along with air and hydrogen through a platinum furnace (300 °C) or by recirculating cyclotron-produced I5O-CO2 through sterile water. However, the need of dynamic imaging for flow qualification and for correction for vascular activity in order to visualize the heart, 15O-water has also been used in humans by administering an inhalation of 15OCO2 which is converted to 15O-water in vivo by the lung enzyme carbonic anhydrase. A recent review provides an excellent summary of these tracers (Schwaiger, 1994). TRACER FOR MEASURING GLUCOSE METABOLISM Under physiological, fasting circumstances, the heart depends on the breakdown of long-chain fatty acids for energy needed for contraction. However, after a carbohydrate meal or with myocardial ischemia, the pattern of substrate use switches to glucose. Although [nC]glucose has been used for assessing myocardial glucose metabolism (Bergmann et al, 1985), FDG, with its 110-minute half-life, has been much more widely available and used for measuring glucose metabolism in man. FDG is a glucose analog with a metabolic pattern different from native glucose. FDG is initially extracted at a rate different than glucose. Once in the myocyte, FDG gets phosphorolated to FDG-6-phosphate. However, this intermediate is not amenable to further glycolytic metabolism. Thus, FDG is a tracer of glucose uptake and differences between its uptake and that of normal glucose need to be accounted for using a correction factor. The switch from fatty acid to glucose metabolism is an important metabolic signature of ischemic myocardium. The use of FDG and a perfusion tracer (usually 13N-ammonia) has become the gold standard for determining ischemic but viable myocardium (Rohatgi et al., 2001; Bax et al., 1997b; Stillman et al., 1999). Extensive efforts in developing single-photon emitting tracers for measuring glucose metabolism have not yielded any promising results. Recently, the use of FDG and single-photon emission computer tomography (SPECT) to detect myocardial viability has been studied and the results are promising (Bax et al., 1997a). If validated in larger numbers of patients, the approach of using positron-emitting tracers with SPECT scanning will widen the application of this approach. FDG is commonly produced by nucleophilic substitution reaction of a O-acetyl-protected mannose triflate with [18F]fluoride (resolubilized in acetonitrile in the presence of K2CO3 and Kryptofix 222) followed by acid hydrolysis. The final tracer is purified using a mixed cation-anion exchange column (Hamacher et al., 1986). RADIOLABELED FATTY ACIDS FOR ASSESSING FATTY ACID METABOLISM The main source of ATP production in normal myocardium is (3-oxidation of fatty acids. Noninvasive assessment of fatty acid metabolism may provide early detection of metabolic abnormalities in the

532

HANDBOOK OF RADIOPHARMACEUTICALS

myocardium (Corbett, 1999). The severity of abnormal cardiac metabolism in patients with acyl-CoA dehydrogenase deficiencies can also be evaluated (Kelly et al., 1993). Many radiolabeled fatty acids have been prepared and evaluated as tracers for measuring cardiac fatty acid metabolism. A. PET tracers [UC]Acetate has been widely used for the noninvasive measurement of myocardial oxygen consumption and for predicting viability (Armbrecht et al., 1989; Brown et al., 1987; Rubin et al., 19%). This 2-carbon fatty acid essentially tracks the activity of the Kreb's cycle. It can be easily prepared by trapping ["CJCOj in an ether solution of ethyl magnesium bromide. However, interpreting changes in myocardial metabolism using ["CJacetate requires concomitant assessment of myocardial perfusion, usually with [15O]water or [13N]ammonia. Since the initial distribution of ["CJacetate is related to blood flow, Gropler et al proposed to use [HC]acetate for indirect measurement of blood flow (Gropler et al., 1991). Recently, Sciacca et al demonstrated that [MC]acetate can be used to quantify myocardial perfusion under resting conditions using a two compartment model without the need for blood sampling (Sciacca et al., 2001). This new approach should increase the usefulness of this tracer and avoid the administration of a separate perfusion tracer. The most commonly used PET ligand for measuring long chain fatty acid metabolism is 1 -[' 'Qpalmitate. 1 ["CJPalmitate is prepared by reacting [nC]CO2 with pentydeconyl magnesium chloride.

l-["C]Palmitate

has been well characterized in both animals and men (Goldstein et al., 1980; Schon et al., 1982). A compartmental modeling technique for quantification of l-["C]palmitate kinetics in nonischemic conditions has been developed (Bergmann et al., 1996). The albumin solution of l-["C]palmitate was prepared by extracting the crude product in ethanol directly with a diluted albumin solution, or by mixing the albumin solution with column purified l-["C]palmitate. Many efforts have been devoted to develop fatty acid analogs that have prolonged retention in the heart. The introduction of a branched methyl group slows the p-oxidation by interfering with the formation of the beta hydroxy CoA intermediate. The steric perturbation introduced by the methyl branches alters the metabolism compared to natural analogs. methylheptadecanoic

acid

Many tracers based on this strategy have been reported.

(1) (Elmaleh

et

al.,

1983;

Livni

et

al.,

1982) and

l-["C]-3l-["C]-3,3-

dimethylheptadecanoic acid (2) (Jones et al., 1988) were developed as metabolically trapped tracer (Figure 2).

These methyl-branched fatty acids were readily prepared by reacting the corresponding Grignard

reagents with [MC]CO2. Recently, [(O-'^jPalmitate was developed as a potential tracer for detecting abnormality of acyl-CoA dehydrogenase deficiencies (Buckman et al., 1994). Since the C-l label of [l-"C)fatty acid is removed in the initial step of fi-oxidation, patients with medium- or short-chain deficiencies would be expected to have normal kinetics in the myocardial handling of l-["C]fatty acid. If (0-["C]palmitate is used, normal metabolism of the tracer will be observed until the tracer is metabolized to a certain chain length which can no longer be handled by the impaired enzyme. Therefore, the ultimate release of o>-f1!C]carbon as ("C]CO2 should be retarded. The prolonged myocardial retention of the extracted w-J1 !C]palmitate may be due to the incorporation of metabolites into amino acids (Buckman et al., 1994). The procedure for the preparation of

RADIOPHARMACEUTICALS FOR STUDYING THE HEART

533

co-[nC]palmitate involved the use of a furanyl Grignard reagent that uses a furan as the masked carboxylic group. After coupling with [HC]methyl iodide, the furan was unmasked using RuC>4. Unfortunately, this procedure is not very reliable due to the need of using large excess of reagents and the difficulties in purification. Recently, two alternative procedures have been proposed. Hosteller et al. used furanyl borane and Pd as catalyst for the [HC]methylation and ozone as the oxidant (Hosteller et al., 1997), and Neu et al. used t-butyl ester of a)-iodocarboxylate and activated Cu for the [nC]methylation and TFA for the deprotection (Neu et al., 1997). The reliabilities of these procedures remain to be demonstrated. Several 118F]-labeled fatty acid derivatives (see Figure 2) have been prepared and evaluated as potential tracers for measuring fatty acid oxidation (De Grado et al., 1991; De Grado et al., 2000; Goodman & Knapp Jr, 1989; Knust et al., 1979; Takahashi et al., 1996; Timothy & Detlef, 1992). Methyl-branched-chain co[!8F]fluorofatty acids have also been reported (Takahashi et al., 1996). Two branched-chain fluorofatty acids, 3-methyl- (3-MFHA) and 5-methyl-17-[18F]fluoroheptadecanoic acid (5-MFHA), have also been prepared. When the initial myocardial uptake was compared, o~['8F]fluoropalrnitic acid (FPA) exhibited the highest uptake, followed by 5MFHA and 3MFHA. FPA has the fastest myocardial washout rate and 3MFHA the slowest. In lipid analysis studies, 5MFHA (similar to FPA) was mainly metabolized to triglyce rides in the heart muscle. However, all three tracers suffered from In vivo defluorination. Recently, several [18F]fluorothiafatty acids were prepared and evaluated (De Grado et al., 1991; De Grado et al., 2000). 14F6THA was shown to be metabolically retained in the myocardium and have excellent imaging properties in normal human subjects and patients with coronary artery disease (Ebert et al., 1994; Maki et al., 1998). However, the tracer failed to track decrease in (3-oxidation in hypoxic conditions (Maki et al., 1998), suggesting that p-oxidation is not solely responsible for intracellular retention of the radiolabel. Prompted by the suboptimal specificity of 14F6THA, several more [18F]-labeled 4- and 6-thia fatty acid analogs were prepared and evaluated (De Grado et al., 2000). Using a perfused heart model, the co-labeled 4thiahexadecanoic acid (FTA) was shown to track the inhibition of oxidation rate of palmitate with hypoxia, whereas that of a 6-thia fatty acid was insensitive to hypoxia. However, in vivo studies showed that FTA underwent defluorination in vivo. The corresponding (o)-3)-[18F]fluoro-4-thia-hexadecanoic acid (13F4THA) showed little in vivo defluorination and is a potential candidate for further evaluation. Among the [ F]-labeled fatty acids, the [ FJfluorothia fatty acids look most promising. If future finding indicates that these tracers can be used for measuring myocardial fatty acid metabolism, the convenient 110min half-life of i8F will enable the distribution of these tracers to PET centers that do not have an in-house cyclotron.

534

HANDBOOK OF RADIOPHARMACEUTICALS CH3-(CH2)14-11CO2H

CH3-(CH2)13-CR2-CH2-11CO2H

[1-11C]Palmitate

R2 = H, CH3 (1) R2 = CH 3 ,CH 3 (2)

11

CH3-(CH2)14-CO2H 11

[co- C]Palmitate

18

F-(CH2)m-S-(CH2)n-C02H

18

F-(CH2)m-CH(CH3)-(CH2)n-CO2H m =12,n

=3 m=14, n = 0

CH3-(CH2)2-C-(CH2)m-S-(CH2)n-C02H

m= 1 1 , n = 4 1 7F6THA (5) m= 12, n = 2 16F4THA (6) m= 6, n = 2 10F4TDA (7)

123

5-FMHA (3) 3-FMHA (4)

m= 7, n = 4 1 4F6THA (8) m= 8, n = 2 13F4THA (9)

(CH2)12-CR2CH2CO2H R2 = H, H R2 = H, CH3 R2 = CH3,CH3

IPPA BMIPP DMIPP

Figure 2. Structures of radiolabeled fatty acids. SPECT tracers. Since the 1960s, iodinated fatty acids have been studied as tracers for measuring myocardial fatty acid metabolism. Two excellent recent reviews detail their development (Corbett, 1999; Knapp Jr. & Kropp, 1999). Initially, straight chain fatty acids, such as 16-I-hexadecanoic acid and 17-1heptadecanoic acid, were introduced. However, the rapid deiodination, short elimination half-time, and the lack of correlation with pVoxidation limited the application of these straight chain fatty acids. To minimize deiodination and slow p-oxidation, iodophenyl fatty acids, such as 15-(para-[l23I]iodophenyl)-pentadecanoic acid (IPPA), 15-p-[123I]iodophenyl-3-(R,S)-methylpentadecanoic acid (BMIPP), and 15-p-[123I]iodophenyl3,3-dimethylpentadecanoic acid (DMIPP), were introduced (see Figure 2) (Knapp Jr et al., 1986; Knapp Jr & Kropp, 1999). Among these three tracers, DMIPP has the slowest myocardial clearance half-time (6 -7 hours), which is followed by BMIPP (30-40 minutes) and IPPA (5-10 minutes). When compared to IPPA, both BMIPP and DMIPP have prolonged myocardial retention with greater accumulations in the subcellular

RADrOPHARMACEUTICALS FOR STUDYING THE HEART

535

microsomal fractions. Currently, BMIPP (Cardiodine) is commercially available in Japan and is the most widely used SPECT tracer for measuring myocardial fatty acid metabolism. Corbett recently reviewed the clinical application of this tracer (Corbett, 1999). TRACERS FOR IMAGING HYPOXIA Most common techniques for the detection of myocardial ischemia involve detection of flow heterogeneity and regional alteration of myocardial metabolism. Although metabolic imaging provides good index of ischemia, the myocardial uptake of metabolic tracers varies widely and depends on flow, the substrate availability and myocardial work, among other reasons. Abnormalities in tissue oxygenation underlie many of the metabolic and contractile changes that occur in ischemic myocardium. [HC]Acetate, as mentioned above, can be used to indirectly assess myocardial oxygen consumption. The use of [15O]oxygen is a more direct way to image myocardial oxygen consumption, but its use is quite complicated and obviously limited to centers with cyclotrons (Yamamoto et al., 1996) Tracers that track low oxygen content would allow tissue hypoxia to be directly assessed, and have the potential to provide a positive image of myocardial hypoxia. An excellent recent review on the potential use of hypoxic markers for myocardial imaging is available (Sinusas, 1999).

H

o

H

NO

HN,/

OH

OH

HL-91

BMS-181321

18

NH(CH2)3F

EF-1

125 i

OH

IV M Figure 3. Molecular structures of radiolabled hypoxic markers.

IAZA

536

HANDBOOK OF RADIOPHARMACEUTICALS

For imaging tissue hypoxia, most of the tracers prepared and evaluated thus far are nitroimidazole based (see Figure 3) (Sinusas, 1999). Nitroimidazoles are a class of lipophilic compounds with high electron affinity that have been developed as radiosensitizers of hypoxic regions in tumors (Chapman, 1979). These compounds readily diffuse through cells. The nitro group can be chemically reduced to form a radical, which under normal oxygen content will react to regenerate the parent compound and diffuse out of the cell. Under hypoxic conditions, the nitro radical can interact with intracellular macromolecules and are trapped (Chapman et al., 1983). Radiolabeled congeners of nitroimidazole were proposed to serve as novel imaging agents for reversibly hypoxic tissue (Shelton et al., 1990; Shelton et al., 1989). The positron-emitting tracer [18F]fluoromisonidazole (FMISO) was shown to have potential for detecting hypoxic myocardium (Martin et al., 1992; Martin et al., 1990; Shelton et al., 1990; Shelton et al., 1989). Shelton et al demonstrated the uptake of FMISO in ischemic and hypoxic hearts was twice that of normal (Shelton et al., 1989). Martin et al. further demonstrated that the myocardial uptake of FMISO is proportional to the level of tissue hypoxia (Martin et al., 1990). Using intact canine model and PET, Shelton et al. demonstrated the increased uptake of FMISO in intact ischemic canine heart (Shelton et al., 1990). Uptake decreased with progressively longer periods of coronary occlusion, and that necrotic myocardium had low uptake. The observation is consistent with the hypothesis that necrotic myocardium has diminished capacity of enzyme for carrying out the nitro reduction. In acute canine models of complete and partial coronary occlusion, Martin et al confirmed the increased uptake in hypoxic myocardium (Martin et al., 1992). FMISO can be easily labeled by reacting 2-nitroimidazole with [l8F]-labeled epifluorohydrin (Grierson et al., 1989; Hwang et al., 1989; McCarthy et al., 1993) or by fluorination of O-THP protected tosylate of misonidazole followed by acidic deprotection (Lim & Berridge, 1993). In addition to FMISO, other fluorinated analogs have been prepared and evaluated for imaging tumor hypoxia. Fluorinated imidazole derivatives, such as fluoroerythronitroimidazole (FETNIM) (Yang et al., 1995) and 2-(2-nitroimidazol-l[H]-yl)-N-(3-fluoropropyl)acetamide (EF1) (Evans et al., 2000), have recently been shown to be potential tracers for hypoxic tumor imaging. Radioiodinated derivatives such as iodoerythronitroimidazole (IETNIM) (Cherif et al., 19%; Inoue et al., 1996), (E)-5-(2-Nitroimidazolyl)-4hydroxy-1-iodopent-l-ene (IVM) (Biskupiak et al., 1991) and l-(5-Iodo-5-deoxy-beta-D-arabinofuranosyl)2-nitroimidazole (IAZA) (Mannan et al., 1991), have also been shown to be potential tracers for hypoxic tumor imaging. They have not been used in imaging myocardial hypoxia. Several Tc-99m labeled nitroimidazoles have been prepared and evaluated as hypoxic imaging agents (see Figure 3). A well-studied tracer is a propylene amine oxime complex of ""Tc, oxo[[33,9,9-tetramethyl-l -(2-nitro-l H-imidazol-1 -yl)4,8-diazaundecane-2,lO
RADIOPHARMACEUTICALS FOR STUDYING THE HEART

537

of BMS-181321 in viable peri-infarct regions was observed, however, the timing of injection relative to the insult and reperfusion was critical (Fukuchi et al., 1996). Using a canine coronary artery occlusion model, BMS-181321 was not retained after a short occlusion time, but increased retention was observed at longer occlusion times (Rumsey et al., 1995a; Rumsey et al., 1995c). A more hydrophilic nitroimidazole derivative of BMS-181321, BMS-194796 (or BRU59-21), has recently been shown to have improved myocardial retention after profound transient ischemia (Rumsey et al., 1995b). Recently, accumulation of BRU59-21 was compared with that of BMS-181321 in Chinese hamster ovary cells incubated under aerobic or hypoxic conditions. The level of selective accumulation in hypoxic cells was 5 times higher than that of aerobic cells. Low levels of oxygen inhibited the maximal accumulation rate by 50%. Unlabeled misonidazole inhibited accumulation of radioactivity, whereas tinidazole, a 5nitroimidazole, enhanced accumulation. These results suggest that BRU59-21 warrants further investigation as an agent for imaging hypoxia (Melo et al., 2000). In addition to nitroimidazole based imaging agent, a recently reported 99mTc compound, 99mTc-HL-91 (HL-91 = 4,9-diaza-3,3,10,10-tetramethyldodecan-2,l 1-dione dioxime), was shown to be a marker of hypoxia and has been suggested as a potential myocardial imaging agent (Okada et al., 1997; Okada et al., 1998). Recently, using isolated heart model, it was shown that nonviable and irreversibly injured myocardium does not take up or retain the tracer (Okada et al., 1999). These initial results demonstrated that 99mTc-HL-91 is a promising tracer for myocardial imaging. One issue that remains to be determined is whether the delivery of tracer to ischemic regions may limit the ability of hypoxic agents to sufficiently sequester in tissue. This will need to be resolved in clinical studies. RADIOTRACERS FOR MYOCARDIAL SYMPATHETIC NERVE IMAGING. Regional and global abnormalities of cardiac sympathetic nervous function are involved in various clinical disorders, such as myocardial infarction, hypertrophic cardiomyopathy, and congestive heart failure. Noninvasive imaging technique provides a method for assessing cardiac sympathetic function in vivo (Glowniak, 1995; Melon & Schwaiger, 1992; Raffel et al., 1995; Schwaiger et al., 1990; Voipio-pulkki, 1995). The major neurotransmitter of the sympathetic nervous system is norepinephrine (NE), which is taken up by the transporter from circulation and stored in neuronal vesicles by vesicular monoamine transporter. Many radiolabeled analogs of NE have been developed to study these transport mechanisms. The most widely used tracer is meta-iodobenzylguanidine (MIBG) (Wieland et al., 1981). MIBG shares cellular transport and storage mechanisms with NE (Figure 4) Both MIBG and NE enter the neuronal cells through the uptake-1 mechanism and are stored in chromaffin granules and secreted in response to acetylcholine. MIBG is rapidly taken up by sympathetic nerves. Using sympathetic denervation models, a profound loss of MIBG uptake in the affected myocardium was observed (Dae et al., 1989; Sisson et al., 1987b). Similarly, a marked loss of MIBG uptake was observed in patients with heart transplants.

538

HANDBOOK OF RADIOPHARMACEUTICALS

Numerous application of MIBG in studying sympathetic innervation and dysfunction of the heart in man have been reported (Dae et al., 1989; Kline et al., 1981; Sisson et al., 1987a; Sisson et al., 1987b; Stanton et al., 1989). A recent review points out the pros and cons of using MIBG to study myocardial sympathetic dysfunction (Glowniak, 1995).

JtL

N

M

NH22 NH

X = I, X X

MIBG

= Br, = •»

Figure 4. Structures of MIBG and derivatives.

With the successful development of MIBG, several analogues of MIBG, such as meta[76Br]bromobenzylguanidine (Dae et al., 1989), para-['8F]fluorobenzylguanidine (Berry et al., 1996), and (4[l8F]fluoro-3-iodobenzyl)guanidine (Vaidyanathan et al., 1994) have been shown to be promising PET tracers for studying sympathetic innervation. Limited information is available regarding these new tracers. Many positron-labeled catecholamine derivatives have been prepared and evaluated (Figure 5) (Raffel et al., 1995). These include ["CJnorepinephrine (Fowler et al., 1974; Nagren et al., 1994), [MC]-metahydroxyephidrine (HED) (Rosenspire et al., 1990), ["CJepinephrine (EPI) (Chakraborty et al., 1993), ["CJmetaraminol (Nagren et al., 1996), and ["CJphenylephrine (PHEN) (Del Rosario et al., 1996). ["CJNorepinephrine and ["C]metaraminol were prepared from ["CJcyanide or ["CJnitromethane (Fowler et al., 1974; Nagren et al., 1994). All other tracers were prepared by ["CJmethylation of the corresponding nor-precursors. All these tracers have been shown to be promising probes for imaging myocardial sympathetic neurotransmission. However, only HED, EPI and PHEN have been evaluated in humans.

RADIOPHARMACEUTICALS FOR STUDYING THE HEART

539

OH

'CJNorepinephrine

[11C]Epinephrine(EPI)

NH11CH3

["C]HED

rC]PHEN

NH2

5 6-['r18 F]fluorodopamine

r18

F]fluoronorepinephrine H

a 6-[r18 F]f luorometaraminol

4-[18F]fluorometaraminol

Figure 5. Structures of positron-labeled norepinephrine and derivatives.

Similar to norepinephrine, HED, EPI and PHEN are transported into storage vesicles by the vesicular monoamine transporter. HED is not a substrate for monoamine oxidase (MAO), but PHEN and EPI lack the methyl group of HED and are substrates of MAO. In isolated rat hearts, the initial uptake rates of PHEN and EPI are considerably lower than that of HED. This difference is mainly due to lipophilicity. Due to efficient vesicular storage, an extremely slow clearance of EPI was observed. When the MAO activity was blocked

540

HANDBOOK OF RADIOPHARMACEUTICALS

with pargyline, a slower clearance rate of PHEN was observed, suggesting MAO activities affect the clearance of PHEN (Raffel & Wieland, 1999). When PHEN and HED are evaluated in normal volunteers, PHEN and HED have similar levels of uptake. However, PHEN has faster efflux rate. It is reasonable to hypothesize that the efflux of PHEN reflects the metabolism of PHEN within the neuron. Currently, HED is the most widely used PET tracer in studying myocardial sympathetic nervous function in humans. However, recent results indicated that PHEN can be a potential tracer for measuring the status of neuronal and vesticular norepinephrine transporters and the oxidative robustness of the neuron (Raffel et al., 1996). Several [18F]-labeled epinephrine derivatives have been prepared and evaluated. Initial efforts focused on radiolabeled metaraminol, which is a false neurotransmitter and has high affinity for the NE transporter and the vesicular monoamine transporter. 6-[l8F]fluorometaraminol was prepared by fluorodemercuration reaction (Mislankar et al., 1988; Rosenspire et al., 1989; Wieland et al., 1990). Despite its good in vivo results and excellent imaging properties, the low specific activity precluded its clinical use. Recently, synthetic methods for the preparation of 6-[l8F]fluorometaraminol and (lR,2S)-4-[18F]fluorometaraminol with high specific activity were reported (Langer et al., 2000). (lR,2S)-4-[18F]fluorometaraminol was shown to be a promising alternative to 6-[l8F]fluorometaraminol for mocardial neuronal mapping with PET. 6-[l8F]fluoronorepinephrine was prepared with high specific activity and both enantiomers were resolved and studied (Ding et al., 1991 a). Comparative PET studies of (-) and (+)-6-[18F)fluoronorepinephrine [(-)-6[18F]FNE and (+)-6-['8F]FNE in the same baboon showed (-)-6-[l8F]FNE is a promising tracer for studying sympathetic functions (Ding et al., 1993). Cardiac uptake of (-)-6-[18F]FNE was greatly reduced when animals were pretreated with desipramine, which interacts with norepinephrine reuptake. The clinical application of this tracer is yet to be demonstrated. In addition to norepinephrine derivatives, l8F-labeled fluorodopamine was also prepared and evaluated (Ding et al., 1993; Ding et al., 1991b; Ding et al., 1995). However, the metabolic profiles of 6[18F]fluorodopamine in the heart were found to be very complicated. Its use remains to be demonstrated. TRACERS FOR IMAGING OTHER MYOCARDIAL RECEPTORS A. p-Adrenergic Receptors. Myocardial adrenergic receptors are involved in many heart diseases, such as ischemia, cardiomyopathy, congestive heart failure, and hypertension. A noninvasive imaging technique to assess the receptor density and drug bindings to these receptors would provide new insights into the involvement of sympathetic nervous system in the etiology of myocardial disease. Several radioligands have been prepared and evaluated to study myocardial adrenoreceptors (Figure 6) These tracers involve both subtype specific and nonspecific ct-adrenergic receptor ligands and (3-adrenergic receptor ligands. A recent review on the selection, design and evaluation of new radioligands for PET studies of cardiac adrenoergic receptors has been published (Pike et al., 2000).

541

A series of fi-blockers have been labeled with positron emitters and evaluated in vivo. These tracers are ["CJpropranolol (Syrota, 1988), [HC]practolol (Syrota, 1988), ["CJatenolol, [uC]metoprolol, [!1C]carazolol (Berridge et al., 1994), [UC]CGP12177 (Hammadi and Crouzel, 1991), and ["C]CGP12388 (Elsinga et al., 1999).

Ar-O OH

H

Ar: NHCOCH3 1

[11C]Practolol

C]Propranoioi

\=/

X

CONH2

[11C]Atenoioi

CjPindolol

-CH2CH2OCH3

[11C]Metoprolol

[11C]Carazolol

1

C]CGP12388

O n 11 CJ HN NH

1

'C]Formoterol

C]CGP12177

C]CGP20712A Figure 6. Structures of radiolabeled a-adrenergic receptor Hgands. Due to difficulty of producing a consistent yield of ["Cjphosgene, which is the precursor for the radiolabeling of CGP12177, several [l!C]-labeled derivatives that can be radiolabeled with [nC]methyl

HANDBOOK OF RADIOPHARMACEUTICALS

542

iodide and [HC]acetone were prepared and evaluated. The [HC]methylated derivative of CGP12177 was found to have high lung uptake and extensive metabolism (Davenport et al., 1995b). More recently, the N[nC]isopropyl derivative of CGP12177, [UC]CGP12388, was prepared and evaluated (Elsinga et al., 1997). In human studies, (S)-[UC]CGP12388 provided good PET images with a specific to non-specific ratio of 4 at 5 min after injection (Elsinga et al., 1999). This uptake is inhibited by pindolol. No radiometabolites were observed with this tracer. The potential use of this promising tracer remains to be demonstrated. Recently, [HC]-labeled formoterol, a & -adrenergic receptor agonist, was prepared and evaluated (Figure 6) (Visser et al., 1998). The biodistribution studies showed significant specific binding in lungs and heart (pV adrenoceptors rich tissues). Binding in these organs was blocked by (32 selective antagonist and not by 3i selective drugs. ["C]Formoterol was rapidly metabolized in rats, but lungs and heart did not substantially take up the labeled metabolites. The ratios of total/nonspecific binding of [nC]formoterol in rat lung reaches only 1.8, which is substantially lower than that of [I!C]CGP12177 (Visser et al., 1998).

18 XCH(CH3)CH2 F

8

F]Fluorometoprolol

18

F]Fluorocarazolol

[18F]FluoroCGP 12388

Figure 7. Molecular structures of l8F-labeled a-adrenergic receptor ligands. Since only an agonist can differentiate the high and low affinity state of G protein coupled ($3 receptors, despite the low total/nonspecific ratio, [ H C] formoterol holds the potential to image the high affinity state of beta2-adrenoceptors. It may provide new insights in the mechanisms underlying prolonged sympathomimetic action. Several [18F] -labeled a-adrenergic receptor ligands have been prepared and evaluated (Figure 7). The most promising tracer is [l8F]fluorocarazolol (Zheng et al., 1994). It has a heart-to-blood ratio of 18 at 45 min after tracer administration, and the ratio decreased to 2 after pindolol administration (Visser et al., 1997). This tracer has also been used to study pulmonary and brain beta adrenergic receptors (van Waarde et al., 1995; van Waarde et al., 1997). Other [l8F]-labeled tracers, such as [l8F]fluorometoprolol and l8 [ F]fluoroCGP12388, have been evaluated. However, [18F]fluorometoprolol had low affinity and [18F]fluoroCGP12388 had high lung uptake.

RADIOPHARMACEUTICALS FOR STUDYING THE HEART

543

One pi selective antagonist, CGP20712A, has been labeled and evaluated (Elsinga et al., 1994). Only modest specific/nonspecific ratio in the rat myocardium was observed with this racemic tracer. The use of the more potent S-entiomer (CGP26505), which might provide a better ratio, is yet to be evaluated. B. a-Adrenergie Receptors. The main a-adrenergic receptor in myocytes is subtype 1 (aj), which is located postsynaptically. This receptor subtype is implicated in arrhythmogenesis, ventricular hypertrophy, and in ischemia. Several PET tracers have been prepared and evaluated (Figure 8). These tracers are [HC]prazosin (Ehrin et al., 1988), [uC]bunazosin (Davenport et al., 1997), and [UC]GB67 (Davenport et al., 1995a). In a canine model, ["Clprazosin was shown to have high nonspecific myocardial uptake. [' 'CjBunazosin has not been well characterized.

CH2CH2CH3

1

[11C]Prazosin

CjBunazosin

[11C]GB67 Figure 8. Structures of [C-l l]-labeled a-adrenorergic receptor ligands. Currently, [UC]GB67 has been used in human studies. GB67 is a potent and selective (Xi adrenergic receptor antagonist (pA2 - 8.93) with moderate lipophilicity (log p = 3). In rodents, a rapid plasma clearan :e of [!1C]GB67 was observed (Law et al., 2000). The maximum myocardial uptake occurred in less than 2 minutes and decreased slowly over 1 hour. Preinjection of GB67 or prazosin (cti antagonist) blocked the myocardial uptake of [UC]GB67. In human studies, high myocardial uptake of the tracer was observed. This tracer is a promising tracer for studying ttj adrenergic receptor in man. C. Muscarinic Receptors. Muscarinic, cholinergic receptors play an important role in the regulation of heart rate and contractile force. Changes in muscarinic receptor density has been associated with various 11

CH<

[11C]MQNB Z-(R,R)-IQNP Figure 9. Molecular structures of radiolabeled muscarinic receptor ligands.

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HANDBOOK OF RADIOPHARMACEUTICALS

physiological, pharmacological, and clinical conditions in animals and humans (Delforge et al., 1990). ["C]-Labeled MQNB, the methylated quaternary ammonium derivative of the potent muscarinic receptor antagonist quinuclidinyl benzylate (QNB), has been evaluated in dogs (Delforge et al., 1990), baboons (Maziere et al., 1981) and in humans (Le Guludec et al., 1997) (Figure 9). The tracer is prepared by reacting ["CJmethyl iodide with QNB to form the methyl ammonium salt of QNB (Maziere et al., 1981). Methods for quantifying the receptor density in humans have been developed (Le Guludec et al., 1997), and an upregulation of myocardial muscarinic receptors has been observed in patients with congestive heart failure. No change of muscarinic receptor densities was observed in heart transplant patients (Le Guludec et al., 1994). More recently, an iodinated tracer, Z-(R,R)-IQNP, was reported to have selective and high uptake in heart and brain (McPherson et al., 2000). demonstrated.

The potential use of this tracer in humans is yet to be

D. Benzodiazepine receptors. The heart is rich in peripheral bezodiazepine receptor (0)3), which is linked to the voltage-operated calcium channels. To date, many benzodiazepine receptor ligands have been radiolabeled and evaluated for imaging central and peripheral bezodiazepine receptors. An excellent review on this subject is available (Pike et al., 1993). Currently, ["C]-labeled PK11195 is the tracer of choice for imaging 0)3 receptor (Figure 10). The tracer is prepared by reacting N-desmethyl-PKl 1195 with [ H C]methyl iodide in the presence of base (Camsonne et al., 1984). [ H C]PK11195 has been used for imaging benzodiazepine receptors in human heart

[11C]PK11195:

R 1 = 1 bH 3 ;

R2 = H;

X = Cl

[18F]PK14105:

R, =

CH3;

R2 = NO2;

X = 18F

CH3;

R2 =H;

X=

[12^]-lodo-PK11195: RI=

123

I

Figure 10. Structures of radiolabeled peripheral benzodiazepine receptor ligands. (Charbonneau et al., 1986). Optically pure enantiomers was observed with the R-isomer, this finding of ["C]PK11195 have been prepared and evaluated (Shah et al., 1994). A slightly higher uptake of receptor rich area, agrees with the fact that the R-isomer is about two-fold higher in affinity. The advantages of using ["C]-(R)-PK11195 for imaging myocardial 0)3 receptors in humans is yet to be demonstrated. In addition to the [C-l l]-labeled PK11195, both [18F]- and [I-123]-labeled PK11195 derivatives are reported. [I8F]PK14105 was prepared by the l8F-for-Cl reaction ontRP58271, the 5-nitro derivative of PK11195 (Pascali et al., 1990). This tracer was found to accumulate in rat striatal lesions, which express the peripheral o>3 receptors (Price et al., 1990). Further studies are needed to demonstrate the use of this tracer for imaging myocardial 0)3 receptors. [ l23 I]-iodo-PKl 1195 has been labeled by an isotopic exchange reaction

RADIOPHARMACEUTICALS FOR STUDYING THE HEART

545

(Gildersleeve et al., 1996). In rodent studies, high uptakes of the tracer were found in peripheral (03 receptor rich tissues and the uptake was blocked by the preadministration of PK11195.

SPECT studies in dogs

showed a high myocardial uptake of the tracer. Thus, this tracer is a promising tracer for SPECT studies of myocardial w$ receptors. FUTURE OUTLOOK Estimates of myocardial perfusion both with SPECT and PET technologies are likely to continue to serve as the mainstay for the diagnosis of coronary artery disease and for evaluation of the efficacy of medical and interventional therapy. With regards to PET tracers, tracers that show good extraction and retention but without background organ uptake and not metabolically linked to metabolism would be valuable. Technetium-based agents will continue to serve as the basis for SPECT flow imaging. Evaluation of myocardial metabolism will continue to be useful not only for assessment of viability, but for also understanding the physiology and pathophysiology of the biochemical reactions that underlie cardiac dysfunction.

Although 1!C-acetate and

18

F-FDG are currently the mainstays of metabolic evaluation of

myocardial viability, a technetium-99m labeled glucose congener with normal metabolism would be of great value. It is estimated that approximately 50% of patients with "scar" by SPECT perfusion imaging have evidence of metabolic viability with PET. It is these patients who are at high risk for cardiac events and who would benefit from revascularization strategies. Direct imaging of hypoxia would be an attractive alternative to metabolic imaging, but the clinical applications of this approach remain to be determined. With the increasing incidence of congestive heart failure in Western society and the role of the neuroendocrine system in the pathophysiology of heart failure, imaging of cardiac neurohormonal function will become increasingly important to the understanding of the development of heart failure and directing pharrnacologic treatment. Finally, although not currently applicable, new genetic therapies such as the administration of angiogenic factor, implantation of cardiac or stem cells into diseased myocardium, or the administration of growth factors into the myocardium are on the horizon. Imaging approaches that permit evaluation of the expression of administered genetic material will likely become increasingly important for cardiac evaluations. In addition, the use of imaging techniques to understand the kinetics of myocardial drug binding and interactions with other drugs and with disease is an untapped technology. Imaging of the heart has contributed greatly to our understanding of the heart. The outlook for future advances continues to be bright.

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Fukuchi K, Kusuoka H, Watanabe Y, Fujiwara T and Nishiniura T (1996) Ischemic and reperfused myocardium detected with technetium-99m-nitroimidazole. J. NucL Med., 37, 761-6. Ghezzi C, Fagret D, Arvieux CC, Mathieu JP, Bontron R, Pasqualini R, de Leiris J and Comet M (1995) Myocardial kinetics of TcN-NOET: a neutral lipophilic complex tracer of regional myocardial blood flow. J. NucL Med., 36,1069-77. Gildersieeve DL, Van Dort ME, Johnson JW, Sherman PS and Wieland DM (1996) Synthesis and evaluation of [l23I]-iodo-PK11195 for mapping peripheral-type benzodiazepine receptors (omega 3) in heart. NucL Med. BioL, 23, 23-8. Glowniak JV (1995) Cardiac studies with metaiodobenzylguanidine: a critique of methods and interpretation of results. J. NucL Med., 36, 2133-7. Goldstein RA, Klein MS, Welch MJ and Sobel BE (1980) External assessment of myocardial metabolism with C-l 1 palmitate in vivo. J. NucL Med., 21, 342-8. Goodman MM and Knapp Jr FF (1989) Radiochemical synthesis of [18F]-3-methyl-branched omega fluorofatty acids. J. Labelled Camp. Radiopharm., 26, 233-235. Gould KL, Goldstein RA, Mullani NA, Kirkeeide RL, Wong WH, Tewson TJ, Berridge MS, Bolomey LA, Hartz RK and Smalling RW (1986) Noninvasive assessment of coronary stenoses by myocardial perfusion imaging during pharmacologic coronary vasodilation. VIII. Clinical feasibility of positron cardiac imaging without a cyclotron using generator-produced rubidium-82. J. Am. Col. Card., 7, 775-89. Green MA, Mathias CJ, Welch MJ, McGuire AH, Perry D, Femandez-Rubio F, Perimutter JS, Raichle ME and Bergmann SR (1990) Copper-62-labeled pyravalofehydebis(N4-n^lhylthioseniicarbazonato)copper(n): synthesis and evaluation as a positron emission tomography tracer for cerebral and myocardial perfusion. J. NucL Med., 31, 1989-%. Grierson JR, Link JM, Mathis CA, Rasey JS and Krohn KA (1989) A radiosynthesis of fluorine-18 fluoromisonidazole. J. NucL Med., 30, 343-50. Gropler RJ, Siegel BA and Geltman EM (1991) Myocardial uptake of carbon-11-acetate as an indirect estimate of regional myocardial blood flow. J. NucL Med., 32, 245-51. Hamacher K, Coenen HH and Stocklin G (1986) Efficient stereospecific synthesis of no-carrier-added 2[18F]-fluoro-2-deoxy-D-glucose using aminopolyether supported nucleophilic substitution. /. NucL Med., 27, 235-8. Hammadi A and Crouzel C (1991) Asymmetric synthesis of (2S)- and (2R)-4-(3-t-butylamino-2-hydroxypropoxy>benzimidazol-2-t1'C]one ((S)- and (R)-[11C]CGP12177) from optically active precursors. J. Labelled Comp. Radiopharm., 29,681-690. Herrero P, Hartman JJ, Green MA, Anderson CJ, Welch MJ, Markham J and Bergmann SR (1996) Regional myocardial perfusion assessed with generator-produced copper-62-PTSM and PET. J. NucL Med., 37, 1294-300. Hosteller ED, Fallis S, McCarthy TJ, Dence CS, Welch MJ and Katzenellenbogen JA (1997) New synthetic routes for the preparation of C-ll palmitic acid labeled at tail positions. J. Labelled Comp. Radiopharm., 40, 779-780. (Abstract)

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Lenihan DJ, McGoron AJ, Gabel M, Walsh RA and Gerson MC (1999) Reliability of technetium-99m Q12 and thallium-201 myocardial activity measurements after triphenyl tetrazolium chloride myocardial staining by perfusion. Investigation Radiology, 34, 276–81. Lim JLand Berridge MS (1993) An efficient radiosynthesis of [18F]fluoromisonidazole. Appl. Rad. Isot., 44, 1085-1091. Livni E, Elmaleh DR, Levy S, Brownell GLand Strauss WH (1982) Beta-methyl[l-11C]heptadecanoic acid: a new myocardial metabolic tracer for positron emission tomography. J. Nucl. Med., 23, 169–75. Luthra SK, Osman S, Steel C, Law M, Rhodes C, Vaja V, Ahier R, Brady F, Waters S, De Silva R, Hughes J and Camici P (1993) Comparison of S-[11C]CGP12177 metabolism in rat, dog and man using solid phase extraction and HPLC. J. Labelled Comp. Radiopharm., 32, 504–505. Maddahi J, Schelbert H, Brunken R and Di Carli M (1994) Role of thallium-201 and PET imaging in evaluation of myocardial viability and management of patients with coronary artery disease and left ventricular dysfunction. J. Nucl. Med., 35, 707–15. Maki MT, Haaparanta M, Nuutila P, Oikonen V, Luotolahti M, Eskola O and Knuuti JM (1998) Free fatty acid uptake in the myocardium and skeletal muscle using fluorine-18-fluoro-6-thia-heptadecanoic acid. J, Nucl. Med., 39, 1320–7. Mannan RH, Somayaji VV, Lee J, Mercer JR, Chapman JD and Wiebe LI (1991) Radioiodinated l-(5-iodo5-deoxy-beta-D-arabinofuranosyl)-2-nitroimidazole (iodoazomycin arabinoside: IAZA): a novel marker of tissue hypoxia. J. Nucl. Med., 32, 1764–70. Martin GV, Caldwell JH, Graham MM, Grierson JR, Kroll K, Cowan MJ, Lewellen TK, Rasey JS, Casciari JJ and Krohn KA (1992) Noninvasive detection of hypoxic myocardium using fluorine-18fluoromisonidazole and positron emission tomography. J. Nucl. Med., 33, 2202–8. Martin GV, Cerqueira MD, Caldwell JH, Rasey JS, Embree Land Krohn KA (1990) Fluoromisonidazole. A metabolic marker of myocyte hypoxia. Clinical Research, 67, 240-4. Maziere M, Comar D, Godot JM, Collard P, Cepeda C and Naquet R (1981) In vivo characterization of myocardium muscarinic receptors by positron emission tomography. Life Science, 29, 2391–7. McCarthy TJ, Dence CS and Welch MJ (1993) Application of microwave heating to the synthesis of [18F]fluoromisonidazole. Appl. Rad. Isot., 44, 1129–1132. MePherson DW, Greenbaum M, Luo H, Beets AL and Knapp Jr FF (2000) Evaluation of Z-(R,R)-IQNP for the potential imaging of m2 mAChR rich regions of the brain and heart. Life Science, 66, 885-96. Melo T, Duncan J, Ballinger JR and Rauth AM (2000) BRU59-21, a second-generation 99mTc-labeled 2nitroimidazole for imaging hypoxia in tumors. J. Nucl. Med., 41, 169–76. Melon PG and Schwaiger M (1992) Imaging of metabolism and autonomic innervation of the heart by positron emission tomography. Euro. J. Nucl. Med., 19, 453–464. Merlet P, Delforge J, Syrota A, Angevin E, Maziere B, Crouzel C, Valette H, Loisance D, Castaigne A and Rande J (1993) Positron emission tomography with [11C]CGP-12177 to assess beta-adrenergic receptor concentration in idiopathic dilated cardiomyopathy [see comments]. Circulation, 87, 1169– 78. Mislankar SG, Gildersleeve DL, Wieland DM, Massin CC, Mulholland GK and Toorongian SA (1988) 6[18F]fluorometaraminol: a radiotracer for in vivo mapping of adrenergic nerves of the heart. J. Med.l Chem., 31, 362–6.

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Nagren K, Schoeps KO, Halldin C, Swahn CG and Farde L (1994) Selective synthesis of racemic l-"C-Iabelled norepinephrine, octopamine, norphenylephrine and phenylethanolamine using [11Clnitromethane. Appl. Rod. Isot., 45, 515–21. Nagren K, Halldin C, Swahn CG, Suhara Tand Farde L (1996) ["CJmetaraminol, a false neurotransmitter preparation, metabolite studies and positron emission tomography examination in monkey. Nucl. Med. BioL, 23, 221–7. Narra RK, Nunn AD, Kuczynski BL, Feld T, Wedeking P and Eckelman WC (1989) A neutral technetium99m complex for myocardial imaging. J. Nucl. Med., 30, 1830–7. Neu H, Kihlberg T and Langstrom B (1997) Synthesis of C-l1-labeled fatty acids using reduced lithium (2thienyl)iodocuprate. J. Labelled Comp. Radiopharm., 40, 796–696 (Abstract) Ng CK, Sinusas AJ, Zaret BL and Soufer R (1995) Kinetic analysis of technetium-99m-labeled nitroimidazole (BMS-181321) as a tracer of myocardial hypoxia. Circulation, 92, 1261–8. Okada RD, Johnson 3rd G, Nguyen KN, Edwards B, Archer CM and Kelly JD (1997) 99mTc-HL91. Effects of low flow and hypoxia on a new ischemia-avid myocardial imaging agent. Circulation, 95, 1892–9. Okada RD, Johnson 3rd G, Nguyen KN, Liu Z, Edwards B, Archer CM, North TL, King AC and Kelly JD (1998) 99mTc-HL91: "hot spot" detection of ischemic myocardium in vivo by gamma camera imaging. Circulation, 97, 2557–66. Okada RD, Johnson 3rd G, Nguyen KN, Carlson LR and Beju D (1999) HL-91-technetium-99m: a new marker of viability in ischemic myocardium. J. Nucl. Card., 6, 306–15. Pascali C, Luthra SK, Pike VW, Price GW, Ahier RG, Hume SP, Myers R, Manjil Land Cremer JE (1990) The radiosynthesis of [18F]PK 14105 as an alternative radioligand for peripheral type benzodiazepine binding sites. Int'l J. Rod. Appl. Instrum. [A], 41, 477-82. Pike VW, Halldin C, Crouzel C, Barre L, Nutt DJ, Osman S, Shah F, Turton DR and Waters SL (1993) Radioligands for PET studies of central benzodiazepine receptors and PK (peripheral benzodiazepine) binding sites—current status [published erratum appears in Nucl. Med. Biol., 1993 Aug; 20(6):I]. Nucl. Med. BioL, 20, 503–25. Pike VW, Law MP, Osman S, Davenport RJ, Rimoldi O, Giardina Dand Camici PG (2000) Selection, design and evaluation of new radioligands for PET studies of cardiac adrenoceptors. Pharm Acta Helv, 74, 191-200. Price GW, Ahier RG, Hume SP, Myers R, Manjil L, Cremer JE, Luthra SK, Pascali C, Pike VW and Frackowiak RS (1990) In vivo binding to peripheral benzodiazepine binding sites in lesioned rat brain: comparison between [3H]PK11195 and [18F]PK 14105 as markers for neuronal damage. J Neurochem, 55, 175–85. Raffel DM, Corbett JR, Schwaiger M and Wieland DM (1995) Mechanism-based strategies for mapping heart sympathetic nerve function. Nucl. Med. Biol., 22, 1019–26. Raffel DM, Corbett JR, del Rosario RB, Gildersleeve DL, Chiao PC, Schwaiger M and Wieland DM (1996) Clinical evaluation of carbon-11-phenylephrine: MAO-sensitive marker of cardiac sympathetic neurons. J. Nucl. Med., 37, 1923–31. Raffel DM and Wieland DM (1999) Influence of vesicular storage and monoamine oxidase activity on ["Cjphenylephrine kinetics: studies in isolated rat heart. J. Nucl. Med., 40, 323–30.

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Rohatgi R, Epstein S, Henriquez J, Ababneh AA, Hickey KT, Pinsky D, Akinboboye O and Bergmann SR (2001) Utility of positron emission tomography in predicting cardiac events and survival in patients with coronary artery disease and severe left ventricular dysfunction. Am. J. Cardio., (in press) Rosenspire KC, Gildersleeve DL, Massin CC, Mislankar SG and Wieland DM (1989) Metabolic fate of the heart agent [18F]6-fluorometaraminol. Int'l. J. Rod. Appl. Instru. B, 16, 735–9. Rosenspire KC, Haka MS, Van Dort ME, Jewett DM, Gildersleeve DL, Schwaiger M and Wieland DM (1990) Synthesis and preliminary evaluation of carbon-11-meta-hydroxyephedrine: a false transmitter agent for heart neuronal imaging. J. Nucl. Med., 31, 1328–34. Rubin PJ, Lee DS, Davila-Roman VG, Geltman EM, Schechtman KB, Bergmann SRand Gropler RJ (1996) Superiority of C-ll acetate compared with 18F fluorodeoxyglucose in predicting myocardial functional recovery by positron emission tomography in patients with acute myocardial infarction. Am.J.CardioL, 78, 1230–5. Rurnsey WL, Kuczynski B, Patel B, Bauer A, Narra RK, Eaton SM, Nunn AD and Strauss HW (1995a) SPECT imaging of ischemic myocardium using a technetium-99m-nitroimidazole ligand. J, Nucl. Med., 36, 1445–50. Rurnsey WL, Patel B and Kuczynski B (1995b) Comparison of two novel technetium agents for imaging ischemic myocardium. Circulation, 92, 1–181. Rurnsey WL, Patel B and Linder KE (1995c) Effect of graded hypoxia on retention of teehnetium-99mnitroheterocycle in perfused rat heart. J. Nucl. Med., 36, 632–6. Sachdev SS, Ramamoorthy N, Nayak UN, Patel RB, Ramanathan P, Srivastava S, Lai R, Raghavan SV, Shah KB and Desai CN (1990) Preparation and evaluation of 99mTc-t-butylisonitrile (99mTc-TBI) for myocardial imaging: a kit for hospital radiopharmacy. Int'l J. Rad. Appl. Instru. B, 17, 543–52, Schon HR, Schelbert HR, Najafi A, Hansen H, Huang H, Barrio J and Phelps ME (1982) C- 11 labeled palmitic acid for the noninvasive evaluation of regional myocardial fatty acid metabolism with positron-computed tomography. II. Kinetics of C- 11 palmitic acid in acutely ischemic myocardium. Am. heart J., 103, 548–61. Schwaiger M, Kalff V, Rosenspire K, Haka M, Molina E, Hutchins GD, Deeb M, Wolfe Jr, E and Wieland DM (1990) Noninvasive evaluation of sympathetic nervous system in human heart by positron emission tomography [see comments]. Circulation, 82, 457-64. Schwaiger M and Muzik O (1991) Assessment of myocardial perfusion by positron emission tomography. Amerocan J. Cardio., 67, 35D–43D. Schwaiger M (1994) Myocardial perfusion imaging with PET. J. Nucl. Med., 35, 693–8. Sciacca RR, Akinboboye O, Chou RL, Epstein S and Bergmann SR (2001) Measurement of myocardial blood flow with PET using l-[11C]acetate. J. Nucl. Med., 42, 63–70. Shah F, Hume SP, Pike VW, Ashworth S and McDermott J (1994) Synthesis of the enantiomers of [Nmethyl-11C]PK 11195 and comparison of their behaviours as radioligands for PK binding sites in rats. Nucl. Med. Biol, 21, 573–81. Shelton ME, Dence CS, Hwang DR, Herrero P, Welch MJ and Bergmann SR (1990) In vivo delineation of myocardial hypoxia during coronary occlusion using fluorine-18 fluoromisonidazole and positron emission tomography: a potential approach for identification of jeopardized myocardium [see comments]. J. Am. Col. Cardio., 16, 477-85.

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Shelton ME, Dence CS, Hwang DR, Welch MJ and Bergmann SR (1989) Myocardial kinetics of fluorine-18 misonidazole: a marker of hypoxic myocardium. J. Nucl. Med., 30, 351-8. Sinusas AJ (1999) The potential for myocardial imaging with hypoxia markers. Semin Nucl Med, 29, 330–8. Sisson JC, Shapiro B, Meyers L, Mallette S, Mangner TJ, Wieland DM, Glowniak JV, Sherman P and Beierwaltes WH (1987a) Meta-iodobenzylguanidine to map scintigraphically the adrenergic nervous system in man. J. Nucl. Med., 28, 1625–36. Sisson JC, Wieland DM, Sherman P, Mangner TJ, Tobes MC and Jacques Jr., S (1987b) Meta-iodobenzylguanidine as an index of the adrenergic nervous system integrity and function. J. Nucl. Med., 28, 1620–4. Stanton MS, Tuli MM, Radtke ML, Heger JJ, Miles WM, Mock BH, Burt RW, Wellman HN and Zipes DP (1989) Regional sympathetic denervation after myocardial infarction in humans detected noninvasively using I-123-metaiodobenzylguanidine. J. Am. Col. Cardio., 14, 1519-26. Stillman AE, Wilke N and Jerosch-Herold M (1999) Myocardial viability. Radial. Clin. No. Am., 37, 361–78, vi. Suzuki K and Yoshida Y (1999) Production of [13N]NH3 with ultra-high specific activity. Appl. Rod. hot., 50, 497-503. Syrota A (1988) Receptor binding studies of the living heart. New Concepts Cardio. Imaging, 4, 141–161. Takahashi T, Nishimura S, Ido T, Ishiwata K and Iwata R (1996) Biological evaluation of 5-methylbranched-chain omega-[18F]fluorofatty acid: a potential myocardial imaging tracer for positron emission tomography. Nucl. Med. Biol., 23, 303–8. Timothy RD and Detlef CM (1992) Non-b-oxidizable (o-[l8F]fluoro long chain fatty acid analogs show cytochrome P-450 mediated defluorination: implication for the design of PET tracers of myocardial

fatty acid utilization. Nucl. Med. Biol., 1 9 , 3 8 9 – 3 analogue for positron emission tomography. J. Med. Chem., 37, 3655–62. Van Waarde A, Elsinga PH, Brodde OE, Visser GM and Vaalburg W (1995) Myocardial and pulmonary uptake of S-l -[18F]fluorocarazolol in intact rats reflects radioligand binding to beta-adrenoceptors. Euro.J. Pharm., 272, 159–68. Van Waarde A, Visser TJ, Elsinga PH, de Jong B, van der Mark TW, Kraan J, Ensing K, Pruim J, Willemsen AT, Brodde OE, Visser GM, Paans AM and Vaalburg W (1997) Imaging beta-adrenoceptors in the human brain with (S)-l-[18F]fluorocarazolol. J. Nucl. Med., 38, 934–9. Vanzetto G, Fagret D, Pasqualini R, Mathieu JP, Chossat F and Machecourt J (2000) Biodistribution, dosimetry and safety of myocardial perfusion imaging agent "TcN-NOET in healthy volunteers. J.

Nucl. Med., 41, 1 4 1 – 8 . V i s s e r TJ, van Waarde A Characterisation of beta2-adrenoceptors, using the agonist [11C]formoterol and positron emission tomography. Euro. J. Pharm., 361, 35–41. Visser TJ, van Waarde A, van der Mark TW, Kraan J, Elsinga P, Pruim J, Ensing K, Jan sen T, Willemsen AT, Franssen EJ, Visser GM, Paans AM and Vaalburg W (1997) Characterization of pulmonary and myocardial beta-adrenoceptors with S-l '-[fluorine-18]fluorocarazolol. J. Nucl. Med., 38, 169–74. Voipio-pulkki L-R (1995) Relevance of PET/SPECT tracers for cardiac neurotransmission. Nucl. Med. Biol., 22, 1027-1035.

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Wieland DM, Brown L, Rogers W, Worthington K, Wu J, Clinthorne N, Otto C, Swanson D and Beierwaltes W (1981) Myocardial imaging with a radioiodinated norepinephrine storage analog. J. Nucl, Med., 22, 22-31. Wieland DM, Rosenspire KC, Hutchins GD, Van Dort M, Rothley JM, Mislankar SG, Lee HT, Massin CC, Gildersleeve DL and Sherman PS (1990) Neuronal mapping of the heart with 6-[18F]fluorometaraminol. J. Med. Chem., 33, 956–64. Yang Dj, Wallace S, Cherif A, Li C, Gretzer MB, Kim EE and Podoloff DA (1995) Development of 18Flabeled fluoroerythronitroimidazole as a PET agent for imaging tumor hypoxia. Radiology, 194, 795800. Yamamoto Y, de Silva R, Rhodes CG, lida H, Lammertsma A A, Jones T and Maseri A (1996) Noninvasive quantification of regional myocardial metabolic rate of oxygen by 15O2 inhalation and positron emission tomography. Experimental validation. Circulation, 94, 808–16. Zheng L, Berridge MS and Ernsberger P (1994) Synthesis, binding properties and 18F- labeling of fluorocarazolol, a high-affinity beta-adrenergic receptor antagonist. J. Med. Chem., 37, 3219–30.

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JOANNA S. FOWLER, NORA D, VOLKOW, YU-SfflN DING, JEAN LOGAN AND GENE-JACK WANG Brookhaven National Laboratory, Upton, NY 1197, USA. INTRODUCTION There is overwhelming evidence that addiction is a disease of the brain (Leshner, 1997). Yet public perception that addiction is a reflection of moral weakness or a lack of willpower persists. The insidious consequence of this perception is that we lose sight of the fact that there are enormous medical consequences of addiction including the fact that a large fraction of the total deaths from cancer and heart disease are caused by smoking addiction. Ironically the medical school that educates physicians in addiction medicine and the cancer hospital that has a smoking cessation clinic are vanishingly rare and efforts at harm reduction are frequently met with a public indignation. Meanwhile the number of people addicted to substances is enormous and increasing particularly the addictions to cigarettes and alcohol. It is particularly tragic that addiction usually begins in adolescence and becomes a chronic relapsing problem and there are basically no completely effective treatments. Clearly we need to understand how drags of abuse affect the brain and we need to be creative in using this information to develop effective treatments. Imaging technologies have played a major role in the conceptualization of addiction as a disease of the brain (Fowler et al, 1998a; Fowler et al., 1999a). New knowledge has been driven by advances in radiotracer design and chemistry and positron emission tomography (PET) instrumentation and the integration of these scientific tools with the tools of biochemistry, pharmacology and medicine. This topic cuts across the medical specialties of neurology, psychiatry, cancer and heart disease because of the high medical, social and economic toll that drugs of abuse, including and especially the legal drugs, cigarettes and alcohol, take on society. In this chapter we will begin by highlighting the important role that chemistry has played in making it possible to quantitatively image the movement of drugs as well as their effects on the human brain. This will be followed by highlights of PET studies of the acute effects of the psychostimulant drugs cocaine and methylphenidate (ritalin) and studies of the chronic effects of cocaine and of tobacco smoke on the human brain. This chapter concludes with the description of a study which uses brain imaging coupled with a specific pharmacological challenge to address the age-old question of why some people who experiment with drugs become addicted while others do not. THE IMPORTANCE OF CHEMISTRY The ability to isolate and monitor specific chemical changes in the human brain and to probe the consequences of chronic drug exposure depends on advances in radiotracer chemistry (Fowler et at., 1997). A broad look at some of the major radiotracers applied to studies of drugs of abuse (Figure i) makes it

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. €2003 John Wilev & Sons. Ltd

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O

CH

N Cl „

HO" \ \ /-\LJ HO-X^^^~-^—"OH

CH,

11

CH3

18p

Cl 18

18

FDG

norchloro-[ F]fluoroepibatidine

11

[ C]L-deprenyl-D2

11

[ C)clorgyline

'CH,

HN

Cl 1

C}cW/weo-methylphenidate

("CJcocaine

["CJ-S-O-raclopride

Figure 1. Structures of some of the radiotracers and labeled drugs used in the study of drugs of abuse.

possible to highlight the fundamental advances upon which they rest. Many of these go back several decades, but they are the workhorses of today's basic and clinical research. LABELED PRECURSORS The excitation function for the 20Ne(d,a)18F reaction coupled with [18F]F2 targets (Lambrecht & Wolf, 1973) and the excitation function for the I8O(p,n)18F reaction (Ruth & Wolf, 1979) coupled with the development of oxygen-18-enriched targets (water and gas) (Firouzbakht et al, 1999) formed the groundwork for almost all of the fluorine-18 labeled tracers in use today (for a review see Kilboum, 1990; Ding & Fowler, 19%). These include 2-deoxy-2-[18F]fluoro-D-glucose (18FDG) (Ido et al., 1978; Hamacher et al., 1986) [18F]fluoroDOPA (Firnau et al., 1986) f18F]receptor and transporter binding tracers (Shiue et al., 1985) and [18F]catecholamines (Ding et al., 1990). All of these reactions have become important scientific tools. The nucleophilic aromatic substitution made it possible to produce F-18 labeled aromatic compounds including those with electron-rich substituents in high specific activity and allowed the first tracer studies of F-18 labeled receptor and transporter binding tracers at subpharmacological doses (Attinaetal., 1983; Ding et al., 1990). The development of gas targets based on the l4N(p,a)11C reaction made the production of carbon-11 relatively straightforward (Christman et al., 1975). Rapid reproducible methods for making ["C]methyl iodide first by the wet chemical methods (Langstrom et al.,1976) and recently by the gas phase method (Larsen et al., 1995; Link et al., 1995) revolutionized tracer development and allowed the synthesis of

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many labeled compounds for measuring drug pharmacokinetics such as [11C]cocaine (Fowler et al, 1989) and [ Cjmethylphenidate (Ding et al., 1994) as well as [ C]raclopride, (Farde et al., 1986) the key radiotracer for measuring dopamine D2 receptor availability and drug-induced changes in dopamine concentration (Volkow et al., 1994). TRACER KINETICS It is widely appreciated that the application of tracer kinetics in the context of specific interventions (pharmacological, isotope, stereochemistry) is a key issue in being able to sensitively measure the availability of a specific molecular target (Mintun et al., 1984). The advantages and limitations of obtaining absolute concentrations of molecular targets and the confounding influence of changes in blood flow on kinetic terms which can be derived from a radiotracer study have generated much research and controversy. The advent of graphical methods first for irreversible systems with the Patlak Plot (Patlak et al., 1983) and then for reversible systems with the Logan Plot (Logan et al,, 1990) has simplified kinetic analysis but at the same time there are pitfalls and each new tracer requires a fresh critical analysis of the factors which contribute to the moment-to-moment changes in radioactivity concentration. It is expected that this subject will continue to lie at the heart of our ability to make a critical assessment of the meaning of measurements. It was also during the last 20 years that measurement of the input function became an integral part of the study and that rapid and sometimes automated methods for making these measures were developed (Alexoff et al., 1995).

MECHANISTIC STUDIES Because PET and single photon emission computed tomography (SPECT) measure photons and not chemical forms, mechanistic approaches are required to attach biochemical significance to the image. Most mechanistic studies are undertaken in small animals which are sacrificed at different times to obtain kinetics, tissue extraction and analysis to assess chemical form and pharmacological blockade to assess specificity. Translational research from animals to humans usually requires assuming that the same issues apply in humans. There are, however, mechanistic tools which have been applied in humans, including stereoselective binding when the tracer or drug contains a chiral center (Fowler et al., 1987; Ding et al., 1997), pharmacological blockade when safety and toxicity permit (Fowler et al., 1996a) the use of deuterium isotope effects (when there is a labile C-H bond in a rate-limiting step) (Fowler et al., 1988, 1995) and labeling in different positions (Halldin et al., 1989; Galley et al., 1994). Additionally other neuroscience tools such as microdialysis have been combined with imaging to assess the relationship between changes in radiotracer concentration as measured by PET and neurotransmitter release (Breier et al., 1997; Dewey et al., 1999). In the near future, the development and use of small animal PET instruments promises to streamline the process and allow imaging of genetically modified animals (Chatziioannou et al., 1999; Jeavons et al., 1999).

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PET STUDIES OF PSYCHOSTIMULANTS COCAINE (-)-Cocaine is a powerful stimulant isolated from erythroxylon coca. It binds to dopamine, norepinephrine and serotonin transporters with micromolar to submicromolar affinity (Ritz et al., 1987). Cocaine's behavioral properties have been generally attributed to its ability to block the dopamine transporter (DAT) causing the elevation of synaptic dopamine (DA) in the nucleus accumbens. Autoradiographic studies of cocaine binding in vitro in human and primate brain sections have been carried out with [3H]cocaine (Biegon et al., 1992) and [3H]WIN 35428 (Madras et al., 1989). The highest density of binding sites for cocaine is in the basal ganglia, the brain region containing the highest density of dopamine terminals, with minimal binding in other brain regions. Similar results were obtained in in vivo studies in the mouse with [3H]WIN 35428 (Scheffel et al., 1991). Cocaine also is a potent local anesthetic (Ritchie & Green, 1990). Though the multiple direct and indirect interactions of cocaine with tissue exists complicating the association of its behavioral and addictive properties with a specific molecular target, there is mounting evidence that the binding of cocaine to the DAT with its rapid ensuing elevation of dopamine dominates its behavioral effects in humans (Volkow et al., 1999b). Cocaine metabolism proceeds by three major initial transformations: cleavage of the benzoyl ester to give ecgonine methyl ester cleavage of the methyl ester to give benzoyl ecgonine and oxidative Ndemethylation to give norcocaine (Kloss et al., 1984). In humans, the predominant pathway is its metabolism by butyrylcholinesterase to form ecgonine methyl ester. The formation of norcocaine which is also pharmacologically active and the only cocaine metabolite which can enter the brain is a minor pathway in humans (Misra et al., 1975) (Figure 2). The behavior of cocaine in the brain and peripheral organs and its effects on peripheral organs have been studied with PET. In addition, the pharmacokinetics of cocaine and another stimulant drug methylphenidate have been compared. BRAIN PHARMACOKINETICS At tracer doses, [N-11C-methyl]cocaine has a very high and rapid uptake and clearance in the human striatum and clearance half-time of about 20 minutes (Fowler et al., 1989). Inhibition of norepinephrine transporter with desipramine did not change [11C]cocaine binding relative to a baseline scan indicating that the contribution of norepinephrine transporters to the PET image is negligible. Though the low specific to non-specific binding ratio of [11C]cocaine as well as its rapid clearance have been limitations to the identification of cocaine binding sites in brain regions other than the striatum, recently [11C]cocaine brain images from 17 normal healthy subjects were averaged to increase the signal-to-noise ratio and to map the binding of this drug in non-striatal areas. Brain regions clustered into high cocaine binding (putamen>accumbens>caudate), moderate binding (thalamus > precuneus > posterior cingulate gyrus > amygdala, hippocampus and temporal pole) and low cocaine binding groups (orbital cortex, precentral gyrus and cerebellum) (Telangero/., 1999).

PET IMAGING STUDIES IN DRUG ABUSE RESEARCH

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+ benzole acid esterases

ecgonlne methyl ester

spontaneous or enzymatic hydrolysis

benzoyl ecgonine

norcocaine

Figure 2. Major metabolic pathways for cocaine.

The temporal relationship between the kinetics of cocaine in the human brain and its behavioral effects was recently studied (Volkow et at., 1997a). In this study the time course for the uptake and clearance of [11C]cocaine co-administered intravenously with behaviorally active doses of unlabeled cocaine was measured in 17 current cocaine abusers. Peak uptake occurred at 5-10 minutes and the half-time of clearance from peak being about 12 minutes (Figure 3). Self-reports of the "high" during the PET study almost perfectly paralleled the time course of cocaine in the striatum for the initial 20 minutes though the high dissipates more rapidly than the levels of [11C]cocaine at later times. The rapid uptake and high concentration of cocaine at dopamine terminals are key contributors to its powerful reinforcing properties. It is well known that the shorter the time between the administration of a drug and its behavioral effects, the more intense the behavioral stimulation (Balster & Schuster, 1973). Cocaine exists in two enantiomers, (-)-cocaine which is the natural product and (+)-cocaine which is produced synthetically. Only (-)-cocaine is behaviorally active. It also has a higher affinity for the DAT (Ritz et al., 1987), a property that has been used to support the hypothesis that there is an association between cocaine's behavioral effects and binding to the DAT (Spealman et al., 1983). However, in PET studies in the baboon with (+)-[11C]-cocaine, no brain uptake was detected due to its very rapid metabolism by plasma butyrylcholinesterase (Gatley et al., 1990, 1991) (Figure 4). Within 30 seconds after injection, (+)-[!1C]cocaine could not be detected in the blood. Hydrolysis was blocked by physostigmine implicating

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100 0>

% peak uptake % peak "high"

80 60 40 20

20

40

60

80

time (min) Figure 3. [11C]Cocaine time course in the striatum of a current cocaine abuser showing the congruence of cocaine binding and the behavioral "high." The dose of unlabeled cocaine (0.6 mg/kg) was coadministered intravenously with [11C]]cocaine (Volkow et al., I997a). For comparison purposes, values are expressed as percent from peak value.

serum butyrylcholinesterase. Comparative studies in vivo and in vitro showed that the hydrolysis of (+)cocaine is at least 1000 times faster than (-)-cocaine. This study revealed for the first time that even though its affinity at the DAT is much lower than that of (-)-cocaine, the lack of brain uptake is an overriding factor in its lack of behavioral activity. In fact (+)-cocaine is a poor choice as a control in behavioral studies. This study illustrates the importance of assessing the metabolism of a drug as well as its receptor binding properties in establishing a mechanistic link between a drug and its behavioral and therapeutic properties. DISTRIBUTION AND KINETICS IN PERIPHERAL ORGANS Cocaine's effects on monoamine concentration in the brain may be mirrored in its effects on monoamine concentration and regulation in the peripheral organs. The short term distribution of [11C]cocaine and its labeled metabolites (at tracer doses) was measured in peripheral organs in 14 healthy male subjects (Volkow et al., 1992b). The rate of uptake and clearance varied with different organs. Peak uptake occurred in heart and kidneys at 2-3 minutes, in the adrenals at 7-9 minutes and in the liver at 10-15

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minutes. There was no uptake in the lungs. The half-times for clearance from the peak uptakes were 10 minutes for the heart and kidneys and 22 minutes for the adrenals. Liver radioactivity plateaued at 10–15 minutes and remained constant for 40 minutes. Though no assessment of the chemical form or binding specificity was made in these studies, the radioactivity in organs with peak uptake at early times (heart, adrenals and kidneys) probably is in the chemical form of cocaine itself while that which slowly accumulates probably reflect labeled metabolites of cocaine.

(-)-[

11

C]cocaine

11

C]cocaine

0

10

20

30 40 50 60 time (minutes)

70

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Figure 4. Time-activity curves for (-)-[''Cjcocaine and (+)-[11C]cocaine in the baboon brain. Note the absence of C-l 1 uptake with (+)-[11C]cocaine due to rapid metabolism by serum butyrylcholinesterase (Galley et al., 1990).

The high uptake of cocaine in the human heart is of potential medical importance because cardiotoxocity is a major medical complication in cocaine abuse (Kloner et al., 1992). Cocaine's peripheral actions involve both direct effects on the myocardium (Polkis et al., 1987) and indirect effects brought about by release of catecholamines from the adrenal glands (Chiueh & Kopin, 1978). The high uptake of cocaine in the heart as demonstrated by PET is consistent with high levels of cocaine in the myocardium of individuals who-

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have died of cocaine overdose (Polkis et al., 1987). The molecular target(s) for cocaine accumulation in the heart were investigated in the baboon (Fowler et al., 1994). Surprisingly, cocaine binding in the heart cannot be blocked by desipramine, tomoxetine, nomifensine, benztropine or cocaine itself. However, cocaine exposure has a prolonged effect on the function of the norepinephrine transporter (the major neuronal element responsible for terminating the action of norepinephrine). PET studies in the baboon and in the dog using 6-[18F]fluoronorepinephrine (Ding et al., 1991) and [11C]hydroxyephedrine respectively have revealed that cocaine blocks norepinephrine transporter function long after the cocaine has cleared from the heart (Fowler et al., 1994; Melon et al., 1994). More specifically, in the baboon heart norepinephrine transporter function is inhibited with only 48% of norepinephrine reuptake recovered by 78 minutes after cocaine administration (Fowler et al., 1994). Similar long term inhibition of norepinephrine transporter function in chronic cocaine users was also evident using [11C]hydroxyephedrine (Melon et al., 1997) suggesting prolonged reduction of norepinephrine reuptake and storage capacity. This may account for some of the reports of cocaine-induced cardiotoxicity in athletes who use cocaine. Exercise would cause a release of norepinephrine which stimulates the adrenergic system. In the normal healthy individual, this would be regulated through the norepinephrine transporter whereas in the cocaine user, this protective mechanism would be disabled.

COMPARATIVE STUDIES OF COCAINE AND METHYLPHENIDATE (RITALIN) Methylphenidate (MP), is a psychostimulant drug used in children for the treatment of Attention Deficit Hyperactivity Disorder (ADHD) (Carrey et al., 1996). The therapeutic properties of MP appear to have been postulated to reside in its ability to block the reuptake of dopamine thereby increasing the concentration of dopamine in the synapse. Methylphenidate is marketed as a mixture of the d-threo and the l-threo enantiomers. Yet the biological activity of these enantiomers is significantly higher than that of the l-threo enantiomer (eudismic ratio-10) (Patrick et al., 1987). Both d-threo and l-rfcreo-methylphenidate were labeled with carbon-11 (Ding et al., 1994) and compared directly in the human brain with PET (Ding et al., 1997). As expected the d-threo enantiomer binds in the striatum while the l-threo enantiomer clears and shows no specific retention. Though MP has been in used for 40 years, it was not until 1999, that the mechanistic framework accounting for why an increase in brain dopamine is therapeutic in the ADHD individual was revealed. SPECT measurements comparing dopamine transporter availability for ADHD adults and age-matched comparison subjects, showed a significant elevation in the DAT in the ADHD subjects (Dougherty et al., 1999; Krause et al., 2000). Because the DAT are responsible for clearing dopamine from the synapse, an excess of the DAT molecules would be predicted to lower synaptic dopamine creating a deficiency which is restored when the DAT is blocked by methylphenidate. The elevation of synaptic dopamine after oral methylphenidate administration has recently been verified in humans (Volkow et al., 2001)

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One of the intriguing issues associated with the therapeutic use of MP, is that it blocks the DAT with a similar affinity to that of cocaine (Volkow et al., 1995). Since blockade of the DAT is associated with the reinforcing effects of cocaine (Ritz et al., 1987) many have expressed concern regarding the potential abuse liability of MP (Parran & Jasinski, 1991). Though MP is self-administered by laboratory animals (Johanson et al., 1975; Bergman et al., 1989) and abused in humans (Parran & Jasinski, 1991), the abuse of MP is much more limited than that of cocaine (National Institute on Drug Abuse, 1995). Also MP abuse occurs predominantly via the intravenous and not the oral route of administration (Parran & Jasinski, 1991). PHARMACOKINETICS In order to understand the similarities and differences between cocaine and methylphenidate, the pharmacokinetics and the degree to which they block DAT at behavioral and therapeutic doses were compared in the human brain with PET. Each drug was labeled with carbon-11. The position of the label in the N-methyl group for cocaine and the O-methyl group for methylphenidate were chosen to remove ambiguity as to whether the labeled drug or its labeled metabolites are visualized in the brain. [The only metabolite of cocaine which can enter the brain is norcocaine (N-demethyl) and this would not be labeled when [N-11C-methyl]cocaine is used. Similarly, ritalinic acid, the major metabolite of methylphenidate would not be labeled and also does not enter the brain.] When administered intravenously, both [ C]cocaine and [ C]methylphenidate reached peak concentration in the brain very rapidly (peak uptake for cocaine was 4-6 minutes and for MP it was 8-10 minutes) when administered intravenously. However, their clearance rates differed markedly; [11C]cocaine clearance was much faster than that of MP (t1/2 = 20 versus 90 minutes). In the case of cocaine, its rapid uptake and clearance paralleled the short-lived, self-reports of "high" induced by the drug (Volkow et al., 1995). In the case of MP, the fast uptake of the drug paralleled only the ascending limb of the time course for the "high", which returned to baseline rapidly even though MP had not cleared from brain (Volkow et al., 1996). The rapid uptake of intravenously administered [11C]cocaine and ["CJmethylphenidate in the brain contrasts to the slow brain uptake of orally administered [11C]methylphenidate which takes about 60 minutes to reach peak uptake in the brain (Volkow et al., 1998) (Figure 5). It has been postulated that this slow brain uptake is the reason why MP does not induce a "high" when administered orally since the rapidity at which drugs of abuse exert their effects has been shown to be crucial in their reinforcing effects (Balsterera/., 1973). DOPAMINE TRANSPORTER OCCUPANCY As part of the investigation into factors which may account for differences between cocaine and methylphenidate, a comparison of their relative occupancies of the DAT at pharmacologically effective doses was also made. Using [11C]cocaine as a tracer for DAT occupancy, it was found that an intravenous dose of 0.075 mg/kg of MP was required to occupy 50% of DAT (Volkow et al., 1996). This is in the same

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range as intravenously administered cocaine which occupies 50% of the DAT at a dose of 0.13 mg/kg. When MP was given orally, a dose of 0.25 mg/kg was required to occupy 50% of the DAT (Volkow et al., 1998) (Figure 6). The latter dose is within the range of therapeutic doses used in the treatment of ADHD. In spite of the fact that oral MP induced levels of DAT blockade similar to those induced by reinforcing doses of cocaine, it did not induce a "high". These comparative PET studies draw a compelling picture of the importance of drug pharmacokinetics and route of administration as major contributing factors in drug abuse versus drug therapy. Indeed, when MP is given intravenously, it also produces a "high" that most cocaine abusers cannot distinguish from that produced by cocaine (Volkow et al., 1996). Such studies measuring pharmacokinetics and DAT occupancy provide important knowledge in the continuing effort to develop a pharmacologic strategy for treating cocaine abuse (O'Brien et al., 1997).

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Figure 5. Time-activity curve for [ C]cocaine in the human striatum after intravenous injection (left panel) compared to the time-activity curve for [11C]methylphenidate administered orally in the baboon striatum (right panel) (Volkow et al., 1997a, 1998). Note that the rate of brain uptake for cocaine administered intravenously is very rapid in contrast to the rate of uptake of methylphenidate administered orally. Though both drugs bind to the dopamine transporter and occupy >50% of the dopamine transporters at behaviorally active doses, it is likely that the difference in rate of uptake between the intravenous and oral routes accounts for the striking different effects of intravenous cocaine and oral methylphenidate.

IMAGING THE COCAINE ABUSER'S BRAIN DOPAMINE FUNCTION IN COCAINE ABUSERS It has been postulated that the chronic blockade of the dopamine transporter by cocaine produces changes in the dopamine system which lead to the compulsive use of the drug. This has been investigated with PET

PET IMAGING STUDIES IN DRUG ABUSE RESEARCH

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Figure 6. Comparison of dopamine transporter occupancy by cocaine administered intravenously and methylphenidate administered orally. Note the similarity between the doses required to occupy 50% of the dopamine transporters for each of the drugs. Dopamine transporter occupancy was measured with ["cjcocaine and PET (Volkow et al., 1997, 1998).

in cocaine abusers using multiple tracers to study glucose metabolism, dopamine D2 receptor availability and dopamine responsivity. In recently detoxified cocaine abusers (< 1 week) brain glucose metabolism was significantly higher in orbitofrontal cortex and in striatum than in healthy non-abusing controls (Volkow et al., 1991). Brain metabolism was highest in subjects tested during the initial 72 hours after withdrawal and cocaine abusers who had the highest metabolic values in orbitofrontal cortex and striatum also had the highest subjective ratings for craving. In contrast, cocaine abusers tested between 1 and 4 months of detoxification showed significant reductions in metabolic activity in prefrontal cortex, orbitofrontal cortex, temporal cortex and cingulate gyrus (Volkow et al., 1992a). F-18-FDG measures brain glucose metabolism which, in part, reflects the energy required to restore membrane potentials (Schwartz et al., 1976). For this reason, regional abnormalities in glucose metabolism

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which are associated with the action of a particular drug can potentially provide mechanistic information on specific neurotransmitters which are associated with these changes. In the case of cocaine, low metabolism in the orbitofrontal cortex and in the cingulate gyms strongly implicates the brain dopamine system since these are the cortical projections of the mid-brain dopamine cell bodies. In order to test the hypothesis that changes in glucose metabolism are associated with the brain dopamine system, dopamine D2 receptor availability was also measured in the same subjects with [18F]N-methylspiroperidol. These studies revealed decreases in dopamine D2 receptor availability in cocaine abusers relative to normal subjects (Volkow et al., 1990). Furthermore levels of dopamine D2 receptors correlated significantly with measures of metabolic activity in orbitofrontal cortex, cingulate gyrus and prefrontal cortex (Volkow et al., 1993). Lower values for D2 receptor concentration were associated with lower metabolism in these brain regions and these reductions in dopamine D2 receptor availability persisted in the follow-up studies performed 3 months after completing the inpatient detoxification program. This observation led to the postulate that DA disruption of the orbitofrontal cortex may be one of the mechanisms underlying the loss of control by the cocaine abuser during a binge or when exposed to cocaine and/or cocaine related cues (Volkow & Fowler, 2000). Thus DA involvement in addiction may be mediated by its interactions with frontal circuits involved in the control of repetitive and impulsive behaviors. Findings from these studies have served to demonstrate that addicted individuals have neurochemical changes in their brains that may underlie their inability to control their impulses to take the drug and may explain relapse. This notion has been reinforced by recent PET studies with [11CJraclopride showing that cocaine abusers when challenged with an acute intravenous dose of the psychostimulant drug methylphenidate, release significantly less dopamine and have a correspondingly lower behavioral response to the drug than normal subjects (Volkow et al., 1997b). This finding supports the postulate that cocaine abusers have decreased dopamine function (Dackis & Gold, 1985). IMAGING THE SMOKER'S BRAIN The effects of nicotine and tobacco smoke on the human brain have been studied using a variety of imaging techniques (Domino, 1995; Stein et al., 1998). Research has focussed almost entirely on nicotine and there is considerable effort currently being devoted to the development of radioligands for imaging brain nicotinic receptors both for studies of addiction and as scientific tools in drug research and development (Ding et al., 2000a-c; Sihver et al., 2000). There is now no doubt that nicotine is the addictive component of cigarette smoke. It is also appreciated that carbon monoxide from smoke is a major players in cardiac disease associated with smoking (Benowitz, 1997) and that tars contain the carcinogens which result in the high incidence of lung and upper airway cancers associated with smoking. However, tobacco smoke contains several thousand chemical compounds and some of these may also contribute to some of its pharmacological effects. Our studies have focussed on the effect of tobacco smoke exposure on the enzyme monoamine oxidase (MAO).

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Monoamine oxidase occurs in most cells in most species (Singer et al., 1995). It is located in the outer mitochondrial membrane and oxidizes amines from both endogenous and exogenous sources. There are two subtypes, MAO A and MAO B which have different substrate and inhibitor specificities. Peripheral MAO plays a protective role in that it breaks down vasoactive amines like tyramine which are present in certain foods and fermented beverages (Anderson et al., 1993). Brain MAO occurs in both neurons and glial cells and plays a role in the regulation of neurotransmitters, the MAO A subtype breaking down norepinephrine and serotonin and the M A O B subtype breaking down phenethylamine. Both subtypes appear to break down dopamine. It has been known for many years that platelet MAO is significantly lower in smokers (Oreland et al., 1981). However, MAO levels are normal in smokers who quit indicating that low MAO B is a pharmacological effect of the smoke rather than a biological characteristic of smokers (Norman et al,, 1987). Similar to the findings of low platelet MAO in smokers, PET studies of normal volunteers with [11CJL-deprenyl-D2 (Fowler et al., 1995) revealed that cigarette smokers had very low brain MAO B while former smokers have normal levels (Fowler et al., 1996b). PET studies measuring MAO A with [11C]clorgyline (Fowler et al., 1987) showed that smokers also have reduced MAO A (Fowler et al., I996a). Inhibition is partial, with average reductions of 30% and 40% being observed for MAO A and B respectively. This observation raises intriguing questions as to whether MAO inhibition by smoke may contribute to some of the behavioral and epidemiological features of smoking including the decreased risk of Parkinson's disease in smokers (Morens et al., 1995) and an increased rate of smoking in depression (Glassman et al., 1990) and in addictions to other substances (Henningfield et al., 1990) and a general prevalence of smoking in psychiatric illnesses (Hughes et al., 1986). Reductions in MAO A and B, in principle, could spare neurotransmitters from oxidation and reduce the production of hydrogen peroxide, a byproduct of MAO catalyzed oxidation (Cohen & Kesler, 1999). MAO inhibition may act synergistically with the dopamine-releasing properties of drugs of abuse by protecting dopamine from metabolism. Nicotine does not inhibit platelet MAO when it is present in the concentrations normally achieved during smoking (Oreland et al., 1981) nor does it inhibit MAO B in the living baboon when administered intravenously (Fowler et al., 1998b). Recently the fractionation of extracts from flue-cured tobacco leaves led to the isolation of a competitive inhibitor of human MAO A (K(i) = 3 pM) and MAO B (K(i) = 6 pM), the structure of which could be assigned as 2,3,6-trimethylbenzoquinone, by classical spectroscopic analysis and confirmed by synthesis (Khalil et al., 2000). This information may help to provide insights into some aspects of the pharmacology and toxicology of tobacco products. While tobacco smokers have an average of 40% lower values of brain MAO B than non-smokers and former smokers, the degree of MAO B inhibition is quite variable between subjects, ranging between 17 and 67%. The variability in the level of inhibition between the smokers was not accounted for by the smoking duration (average 24 ± 13.5 years) or the frequency (average 1 ± 0.27 packs/day). Because the

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time interval between the PET MAO B measurements and the last cigarette varied between subjects (range 1.7-12 hours), a study was undertaken to determine whether MAO B activity recovered measurably after an overnight smoke-free interval (Fowler et al., 2000). Brain MAO B was measured using PET and [11C]Ldeprenyl-D2 in six smokers who were scanned twice, once at 11.3 hours (baseline) after last cigarette and once at 10 minutes after smoking. Brain MAO B levels did not differ between baseline and after smoking as determined using a three compartment model (Logan et al., 2000) (Figure 7A).

Figure 7. Bar graphs showing that smoking a single cigarette does not produce a measureable inhibition of MAO B in different brain regions (left panel) and that an overnight cigarette abstinence does not lead to a measureable recovery of MAO B (right panel). These two studies were done in non-smokers (Fowler et al., 1999b) and smokers (Fowler et al., 2000) respectively. These studies show that brain MAO B inhibition in smokers requires chronic exposure and that the recovery of brain MAO B is slow. Studies were done with [11C]L-deprenyl-D2 and PET

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Another aspect of the pharmacodynamic relationship between tobacco smoke exposure and MAO inhibition relates to whether MAO inhibition can be detected after a single cigarette. For this purpose brain MAO B was measured in a group of 8 non-smokers at baseline and immediately after smoking a single cigarette using [11C]L-deprenyl-D2 and PET. Eight normal healthy non-smokers (35 ± 11 years) received two PET studies 2 hours apart with [11C]L-deprenyl-D2, one at baseline and the second 5-10 minutes after the subject has smoked a single cigarette (Fowler et al., 1999b). Plasma nicotine and expired carbon monoxide (CO) were measured prior to smoking and 10 minutes after smoking completion as an index of tobacco smoke exposure. A 3-compartment model was used to calculate Ak3, a model term proportional to MAOB and K; for the plasma-to-brain transfer constant which is related to brain blood flow. The average 1A; and K1, for different brain regions did not differ significantly between baseline and smoking (Figure 7B). These results indicate that the reduction in MAO B in smokers occurs gradually and requires chronic tobacco smoke exposure. The observation that smokers have reduced brain MAO A and B reinforces the importance of reporting smoking status in clinical studies and the need to reevaluate reports that low platelet MAO B is a biological marker in clinical populations where the rate of smoking is high such as schizophrenia (Lidberg et al., 1985). In fact, normal platelet MAO was recently reported in non-smoking patients with schizophrenia (Simpsons al., 1999). Smoking remains a major public health problem. Yet advances in treating smoking addiction hinge on characterizing both the neuropharmacological effects of tobacco smoke and factors accounting for individual variability in smoking toxicity. Along this line recent studies reporting the use of the reversible MAO A inhibitor moclobemide (Berlin et al.,1995a,b) and the combination of nicotine and L-deprenyl (Brauer et al., 2000) as smoking cessation treatments is an important step based on the knowledge that the addictive and toxic effects of tobacco smoke are complex and go beyond the effects of nicotine alone. VULNERABILITY The questions of why some people who experiment with drugs become addicted while others do not is an intriguing one. One of the hypotheses is that there are individual genetic factors which make some individuals more vulnerable to addiction. The "reward deficiency hypothesis" postulates that addictive behaviors, both pharmacological and non-pharmacological (gambling, for example) emerge as a result of understimulation of reward circuits with the drug taking or other behavior being used to stimulate these reward circuits. Indeed a variant on the dopamine D2 receptor (the Taql A1, allele) has been reported to occur more frequently in individuals with abnormal appetitive behaviors (Blum et al., 1996).

During our measures of dopamine receptor availability in cocaine abusers and in comparison subjects it was evident that there is enormous variability among individuals. Though cocaine abusers as a group have

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significant decrements in dopamine D2 receptors, there is considerable overlap in values with normal individuals (Volkow et al., 19995). The great variability among individuals inspired a study designed to examine whether individuals with low dopamine receptor availability would react differently to a challenge with a stimulant drug (methylphenidate) than individuals with high dopamine receptor availability. It was predicted that individuals with low dopamine receptor levels would find methylphenidate more pleasurable than those with high receptor levels. This proved to be the case. Low receptor level individuals found methylphenidate pleasant while, on average, high receptor level individuals found it unpleasant (Figure 8). This supports the notion that individuals with low dopamine receptors may have an understimulated reward system and as a result they perceive a pleasurable sensation when subjected to a drug-induced elevation in dopamine. It follows that if an individual who takes a drug and finds it pleasant is more likely to repeat the behavior.

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Figure 8. Comparison of dopamine D2 receptor levels in normal subjects who were also tested with a drug challenge of intravenous methylphenidate. On average, those who found methylphenidate pleasant had lower dopamine D2 receptor levels and those who found it unpleasant had higher dopamine receptor levels. Two of the subjects had a neutral response to the drug. Dopamine D2 receptor levels were measured with [11C]raclopride and PET (Volkow et al., 1999b).

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SUMMARY Over the past 25 years radiotracers for many of the major molecular targets of drugs of abuse have been synthesized, characterized pharmacologically, their kinetics have been modeled and they have been used to address medical questions directly in the relevant clinical population. Already neuroimaging studies of the pharrnacokinetics and pharmaocdynamics of abused substances have started to document mechanisms of reinforcement and to delineate neurochemical changes in the brain of the addicted subject. Though these findings are still of a preliminary nature they indicate that imaging has enormous value in the area of substance abuse including: (1) the direct assessment of the behavior of drugs of abuse in the human brain. This is relevant both because drug pharmacokinetics and pharmacodynamics may vary across animal species. It also enables the assessment of drug behavior directly in the drug addict; (2) Because imaging studies are done in awake human subjects, PET imaging allows a determination of the relation between behavior and regional brain effects both in neurotransmitters as well as in function as assessed with measures of glucose metabolism or cerebral blood flow. Studies can also be done to assess the relation between pharmacokinetics of a given drug and the time course of its pharmacological effects; (3) Serial imaging studies can be performed in the same subject allowing an evaluation of the effects of drug withdrawal or drug treatment; (4) Neurochemical and functional changes can be viewed from many perspectives directly in the addicted individual; (5) Application of new knowledge in the development and assessment of new therapeutic interventions. ACKNOWLEDGMENT This research was performed in part at Brookhaven National Laboratory under contract DE-AC0298CH10886 with the U. S. Department of Energy and was supported by its Office of Biological and Environmental Research and by the National Institutes of Health (NS 15380). The authors also thank David Schlyer for his valuable comments. REFERENCES Alexoff DL, Shea C, Fowler JS, King P, Gatley SJ, Schlyer DJ and Wolf AP (1995) Plasma input function determination for PET using a commercial laboratory robot. Nucl. Med. & Biol., 22, 893–904. Anderson MC, Hasan F, McCrodden JF and Tipton KF (1993) Monoamine oxidase inhibitors and the cheese effect. Neurochemical Research, 18, 1145–1149. Attina M, Cacace F and Wolf AP (1983) Displacement of nitro group by 18F-fluoride ion. A new route to high specific activity aryl fluorides. J. Chem. Soc., Chem. Comm,, 107–109. Balster RL and Schuster CR (1973) Fixed interval schedule of cocaine reinforcement: Effect of dose and infusion duration. J. Exp. Analyt. Behav., 20; 119–129. Benowitz NL (1997) The role of nicotine in smoking-related cardiovascular disease. Preventive Medicine 26, 412–417.

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Bergman J, Madras B, Johnson SE and Spealman RD (1989) Effects of cocaine and related drugs in nonhuman primates. HI. Self administration by squirrel monkeys. J. Pharm. Exper. Ther., 251, 150-155. Berlin I, Said S, Spreux Varoquaux O, Launay JM, Olivares R, Millet V, Lecrubier Y and Puech AJ (1995a) A reversible monoamine oxidase A inhibitor (moclobemide) facilitates smoking cessation and abstinence in heavy, dependent smokers. Clin. Pharm.& Ther., 58, (4), 444–52. Berlin I, Said S, Spreux-Varocuax O, Olivares R, Launay J-M and Puech A (1995b) Monoamine oxidase A and B in heavy smokers. Biological Psychiatry, 33, 756–761. Biegon A, Dillon K, Volkow ND, Hitzemann RJ, Fowler JS and Wolf AP (1992) Quantitative autoradiography of cocaine binding sites in human brain postmortem. Synapse, 10, 126–130. Blum K, Cull JG, Braverman ER and Comings DE (1996) Reward deficiency syndrome. Am. Sci. 84, 145. Brauer LH, Paxton DA and Rose JE (2000) Selegiline and transdermal nicotine for smoking cessation. Presented at the 6th Annual Meeting of the Society for Research on Nicotine and Tobacco, February 18-20, Arlington, VA. Breier A, Su TP, Saunders R, Carson RE, Kolachana BS, de Bartolomeis A, Weinberger DR, Weisenfeld N, Malhotra AK, Eckelman WC and Pickar D (1997) Schizophrenia is associated with elevated amphetamine-induced synaptic dopamine concentrations: evidence from a novel positron emission tomography method. Proceedings of the National Academy of Science USA, 94, 2569-2574. Carrey NJ, Wiggins DM and Milin RP (1996) Pharmacological treatment of psychiatric disorders in children and adolescents: focus on guidelines for the primary care practitioner. Drugs, 51, 750-759. Chatziioannou AF, Cherry SR, Shao Y, Silverman RW, Meadors K, Farquhar TH, Pedarsani M and Phelps ME (1999) Performance evaluation of microPET: A high-resolution lutetium oxyorthosilicate PET scanner for animal imaging. J. Nucl. Med,. 40, 1164–1175. Chiueh CC and Kopin IJ (1978) Centrally mediated release by cocaine of endogenous epinephrine and norepinephrine from the sympathoadrenal medullary system of unanesthetized rats. J. Pharm. Exp.l Ther,. 205, 148–154. Christman DR, Finn RD, Karlstrom KI and Wolf AP (1975) The production of ultra high activity "Clabeled hydrogen cyanide, carbon dioxide, carbon monoxide, and methane via the l4N(p,a)11C reaction. XV. Int'lJ. Appl. Rad. Isot. 26, 435-442. Cohen G and Kesler N (1999) Monoamine oxidase and mitochondrial respiration. J. Neurochem., 73, 2310-2315. Dachis CA and Gold MS (1985) New concepts in cocaine addiction: the dopamine depletion hypothesis. Neuroscience and Biobehavioral Review 9, 469–477. Dewey SL, Brodie JD, Gerasimov M, Horan B, Gardner EL and Ashby CR, Jr. (1999) A pharmacologic strategy for the treatment of nicotine addiction. Synapse, 31, 76-86. Ding Y-S, Fowler JS, Gatley SJ, Dewey SL, and Wolf AP (1991) Synthesis of high specific activity (+) and (-)- 6-[l8F]fluoronorepinephrine via the nucleophilic aromatic substitution reaction. J. Med. Chem., 34,767-771.

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Ding Y-S, Sugano Y, Fowler JS and Salata C (1994) Synthesis of the racemate and individual enantiomers of [11C]methylphenidate for studying presynaptic dopaminergic neurons and positron emission tomography. J. Labell. Comp. & Radiopharm., 34, 989-997. Ding Y-S, Shiue C-Y, Fowler JS, Wolf AP and Plenevaux A (1990) No-carrier-added (NCA) aryl[18F]fluorides via the nucleophilic aromatic substitution of electron rich aromatic rings. J. Fluorine Chem., 48, 189–205. Ding Y-S and Fowler JS (1996)

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F- Labeled tracers for positron emission tomography studies in the

neurosciences. In Biomedical Frontiers of Fluorine Chemistry, Chapter 23, 328-343. Ding Y-S, Fowler JS, Volkow ND, Dewey SL, Wang G-J, Gatley J, Logan J and Pappas N (1997) Chiral drugs: Comparison of the pharmacokinetics of [ ll C]d-threo and l-threo-methylphenidate in the human and baboon brain. Psychopharmacology, 131, 71–78. Ding Y-S, Logan J, Beimel R, Garza V, Rice O, Fowler JS and Volkow ND (2000a) Dopamine receptor-mediated regulation of striatal cholinergic activity: PET studies with [!8F]norchlorofluoroepibatidine. J. Neurochem.,,74, 1514–1521. Ding Y-S, Volkow ND, Logan J, Garza V, Pappas N, King P and Fowler JS (2000b) Occupancy of brain nicotinic acetylcholine receptors by nicotine doses equivalent to those obtained when smoking a cigarette. Synapse, 35, 234-237. Ding Y-S, Liu N, Wang T, Marecek J, Garza V, Ojima I and Fowler JS (2000c) Synthesis and evaluation of 6[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine as a PET tracer for nicotinic acetylcholine receptors. Nucl. Med.&Biol., 27, 381–389. Domino EF (1995) Brain Imaging of Nicotine and Tobacco Smoking. NPP Books, Ann Arbor, Michigan. Dougherty DD, Bonab AA, Spencer TJ, Rauch SL, Madras BK, and Fischman AJ (1999) Dopamine transporter density in patients with attention deficit hyperactivity disorder. Lancet, 354, 2132–2133. Farde L, Ehrin E, Eriksson L, Greitz T, Hall H, Hedstrom CG, Litton JE and Sedvall G (1985) Substituted benzamides as ligands for visualization of dopamine receptor binding in the human brain by positron emission tomography. Proceedings of the National Academy of Science USA, 82, 38633867. Firnau G, Garnett ES, Chirakal R, Sood S, Nahmias C and Schrobilgen G (1986) [18F]Fluoro-L-DOPA for the in vivo study of intracerebral dopamine App. Rad. Isot. 37, 669–675. Firouzbakht ML, Schlyer DJ and Fowler JS (1999) Cryogenic target design considerations for the production of [18F] fluoride from enriched [18O]carbon dioxide. Nucl. Med. & Biol., 26, 749-753. Fowler JS, MacGregor RR, Wolf AP, Arnett CD, Dewey SL, Schlyer D, Christman D, Logan J, Smith M, Sachs H, Aquilonius SM, Bjurling P, Halldin C, Hartwig P, Leenders KL, Lundquist H, Oreland L, Stalnacke C-G and Langstrom B (1987) Mapping human brain monoamine oxidase A and B with ''C-suicide inactivators and positron emission tomography. Science, 235, 481-485. Fowler JS, Wolf AP, MacGregor RR, Dewey SL, Logan J, Schlyer DJ and Langstrom B (1988) Mechanistic positron emission tomography studies. Demonstration of a deuterium isotope effect in the MAO catalyzed binding of [11C]L-deprenyl in living baboon brain. J. Neurochem. 51, 15241534.

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Fowler JS, Volkow ND, Wolf AP, Dewey SL, Schlyer DJ, MacGregor RR, Hitzemann R, Logan J, Bendriem B, Gatiey SJ and Christman D (1989) Mapping cocaine binding in human and baboon brain in vivo. Synapse, 4, 371-377. Fowler JS, Ding Y-S, Volkow ND, Martin T, MacGregor RR, Dewey S, King P, Pappas N, Alexoff D, Shea C, Gatiey SJ, Schlyer DJ and Wolf AP (1994) PET studies of cocaine inhibition of the myocardial norepinephrine uptake. Synapse 16, 312-317. Fowler JS, Wang G-J, Logan J, Xie S, Volkow ND, MacGregor RR, Schlyer DJ, Pappas N, Alexoff DL, Patlak C and Wolf AP (1995) Selective reduction of radiotracer trapping by deuterium substitution: comparison of ["C]L-deprenyl and ["C]L-deprenyl-D2 for MAO B mapping. J. Nucl. Med. 36, 1255-1262. Fowler JS, Volkow ND, Wang G-J, Pappas N, Logan J, Shea C, Alexoff DL, MacGregor RR, Schyler DJ, Wolf AP and Zelzukova I (1996a) Brain monoamine oxidase a inhibition in cigarette smokers. Proceedings of the National Academy of Science USA, 93, 14065-14069. Fowler JS, Wang G-J, Volkow ND, Pappas N, Logan J, MacGregor RR, Alexoff D, Wolf AP, Warner D, Cilento R and Zezulkova I (1996b) Inhibition of monoamine oxidase B in the brains of smokers. Nature, 379, 733-736. Fowler JS and Wolf AP (1997) Working against time: Rapid radiotracer synthesis and imaging the human brain. Accounts of Chemical Research, 30,181-188 Fowler JS and Volkow ND (1998a) PET imaging studies in drug abuse. Clinical Toxicology, 36, 163-174. Fowler JS, Volkow ND, Logan J, Pappas N, King P, MacGregor R, Shea C, Garza V and Gatiey SJ (1998b) An acute dose of nicotine does not inhibit MAO B in baboon brain in vivo. Life Science 63(2), PL 19-23. Fowler JS, Volkow ND, Wang G-J, Ding Y-S and Dewey SL (1999a) PET and drug research and development. J. Nucl. Med., 40, 1154-1163. Fowler JS, Wang G-J, Volkow ND, Franceschi D, Logan J, Pappas N, Shea C, MacGregor RR and Garza V (1999b) Smoking a single cigarette does not produce a measurable reduction in brain MAO B in non-smokers. Nicotine and Tobacco Research, 1, 325-329. Fowler JS, Wang G-J, Volkow ND, Franceschi D, Logan J, Pappas N, Shea C, MacGregor RR and Garza V (2000) Maintenance of MAO B inhibition in smokers after a 12-hour cigarette abstinence. Am. J. Psych., 157,1864-1866. Gatiey SJ, MacGregor RR, Fowler JS, Wolf AP, Dewey SL and Schlyer DJ (1990) Rapid stereoselective hydrolysis of (-f)-cocaine in baboon plasma prevents its uptake in the brain: Implications for behavioral studies. J. Neurochem., 54, 720-723. Gatiey SJ. (1991) Activities of the enantiomers of cocaine and some related compounds as substrates and inhibitors of plasma butyrylcholinesterase. Biochemical Pharmacology, 41, 1249-1254. Gatiey SJ, Yu D-W, Fowler JS, MacGregor RR, Schlyer DJ, Dewey SL, Wolf AP, Martin T, Shea CE and Volkow ND (1994) Benzoylecgonine, and C-l 1 and F-18 4-fluorococaine, to probe the extent to

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which C-l 1 cocaine metabolites contribute to PET images of the baboon brain. J. Neurochem., 62, 1154-1162. Glassman AH, Helzer JE, Covey LS, Cottier LB, Stetner F, Tipp JE and Johnson J (1990) Smoking, smoking cessation, and major depression. J. Am. Med. Assoc., 264,1546-1549. Halldin C, Burling P, Stalnacke C-G, Jossan SS, Oreland L and Langstrom B (1989) 1!C-Labeling of dimethylphenethylamine in two different positions and biodistribution studies. Appl. Rod, & /sof.,40, 557-560, Hamacher K, Coenen HH, Stocklin G (1986) Efficient stereospecific synthesis of NCA 2-[18F]-fluoro~2~ deoxy-D-glucose using aminopolyether supported direct nucleophilic substitution. J. Nucl. Med., 27, 235-238. Henningfield JE, Clayton R and Pollen W (1990) Involvement of tobacco in alcoholism and illicit drug use. Brit. J. Addiction, 85, 279-292. Hughes JR, Hatsukami DK, Mitchell JE and Dahlgren LA (1986) Prevalence of smoking among psychiatric outpatients. Am. J. Psych., 143, 993-997. Ido T.Wan C-N, Casella V, Fowler JS, Wolf AP, Reivich M and Kuhl DE (1978) Labeled 2-deoxy-Dglucose analogs. l8F-labeled 2-deoxy-2-fluoro-D-glucose, 2-deoxy-2-fluoro-D-mannose and C-l 4-2deoxy-2-fluoro-D-glucose. J. Labell. Comp. Radiopharm. 14, 175-182. Jeavons AP, Chandler RA, and Dettmar CAR (1999) A 3D HID AC-PET camera with sub-millimetre resolution for imaging small animals. IEEE Trans. Nucl. Sci. 46, 468-473. Johanson CE and Shuster CR (1975) A choice procedure for drug reinforcers: cocaine and methylphenidate in the rhesus monkey. J. Pharm. Exp. Ther., 193, 676-688. Khalil AA, Steyn S and Castagnoli N (2000) Isolation and characterization of a monoamine oxidase inhibitor from tobacco leaves. Chem. Res. Tax. 13, 31-35. Kilbourn MR (1990) Fluorine-18 Labeling of Radiopharmaceuticals. National Academy Press, Washington D. C., 149 Kloner RA, Hale RS, Alker K and Rezkalla S (1992) The effects of acute and chronic cocaine use on the heart. Circulation ,85, 407-419. Kloss MW, Rosen GM and and Rauckman EJ (1984) Cocaine-mediated hepatotoxicity: A critical review. Biochem. Pharm., 33, 169. Krause K, Dresel SH, Krause J, Kung HF and Tatsch K (2000) Increased striatal dopamine transporter in adult patients with attention deficit hyperactivity disorder: effects of methylphenidate as measured by single photon emission computed tomography. Neuroscience Letter, 285, 107-110. Lambrecht RM and Wolf AP (1973) Cyclotron and short-lived halogen isotopes for radiopharmaceutical applications. Proceedings of the IAEA Symposium "Radiopharmaceuticals and Labelled Compounds", 1,275-290. Langstrom B and Lundqvist H (1976) The preparation of "C-methyl iodide and its use in the synthesis of 1 'C-methyl-L-methionine. Int'l J. Appl. Rod. hot.,21, 357-363. Larsen P, Ulin J and Dahlstrom K (1995) A new method for production of nC-labeled methyl iodide from u C-methane. J. Labell. Comp. Radiopharm.., 37, 73-75.

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Leshner Al (1997) Addiction is a brain disease and it matters. Science, 278, 45-47. Lidberg L, Modin I, Oreland L, Tuck JR and Gillner A (1985) Platelet monoamine oxidase activity and psychopathy. Psychiatry Research, 4, 339-43. Link JM, Clark JC, Larsen P and Krohn K (1995) Production of [HC]methyl iodide by reaction of "CH4 with I2. J. Labell. Comp. Radiopharm.., 37, 76-78. Logan J, Fowler JS, Volkow ND, Wolf AP, Dewey SL, Schlyer DJ, MacGregor RR, Hitzemann R, Bendriem B, Gatley SJ and Christman DR (1990) Graphical analysis of reversible radioligand binding from time-activity measurements applied to [N-"C-methyl]-(-)-cocaine PET studies in human subjects. J. Cereb. Blood Flow & Metab., 10, 740-747. Logan J, Fowler JS, Volkow ND, Wang G-J, MacGregor RR and Shea C (2000) Reproducibility of repeated measures of deuterium substituted [MC]L-deprenyl (["C]L-deprenyl-D2) binding in the human brain. Nucl. Med. & Biol.,21', 43-49. Madras BK, Spealman RD, Fahey MA, Neumeyer JL, Saha JK and Milius RA (1989) Cocaine receptors labeled by [3H]2p-carbomethoxy-3p-(4-fluorophenyl)tropane. Mol. Pharm. 36, 518-524. Melon PG, Ngyyen N, DeGrado T, Manger TJ, Wieldand DM and Schwaiger M (1994) Imaging of cardiac neuronal function after cocaine exposure using carbon-11 hydroxyephedrine and positron emission tomography. J. Am. Col. Card., 23, 1693-1699. Melon PG, Boyd CJ, McVey S, Manger TJ, Wieland DM and Schwaiger M (1997) Effects of active cocaine use of cardiac sympathetic neuronal function assessed by carbon-11 hydorxyephedrine. J. Nucl. M**/., 38, 451-456. Mintun MA, Raichle ME, Kilbourn MR, Wooten GF and Welch MJ (1984) A quantitative model for the in vivo assessment of drug binding sites with positron emission tomography. Annals of Neurology, 15, 217-227. Misra AL, Nayak PK, Bloch R and Mule SJ (1975) Estimation and disposition of [3H]benzoylecgonine and pharmacological activity of some cocaine metabolites. J. Pharmaceutical Pharmacol., 27, 784-786. Morens DM, Grandinetti A, Reed D, White LR and Ross GW (1995) Cigarette smoking and protection from Parkinson's disease: false association or etiological clue. Neurology, 45, 1041-1051. National Institute on Drug Abuse, Community Epidemiology Work Group (CEWG) (1995) Epidemiologic Trends in Drug Abuse. DHHS Pub. No. (NIH) 95-3988. Norman TR, Chamberlain KG and French MA (1987) Platelet monoamine oxidase: low activity in cigarette smokers. Psychiatry Research, 20, 199-205. O'Brien CP (1997) A range of research-based pharmacotherapies for addiction. Science, 278, 66-70. Oreland L, Fowler CJ and Schalling D (1981) Low platelet monoamine oxidase activity in cigarette smokers. Life Science, 29, 2511 -2518. Parran TV and Jasinski DR (1991) Intravenous methylphenidate abuse: prototype for prescription drug abuse. Arc. Internal Med., 151,781-783. Patlak C, Fenstermacher JD and Blasberg RG (1983) Graphical evaluation of blood-to-brain transfer constants from multiple time-activity data. J. Cereb. Blood Flow & Metab., 3, 1 -7.

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Patrick KS, Caldwell RW, Ferris RM and Breese GR (1987) Pharmacology of the enantiomers of threomethylphenidate. J. Pharm. Exp. Ther., 241, 152-8 Polkis A, Maginn D and Barr JL (1987) Tissue disposition of cocaine in man: A report of five fatal poisonings. Forensic Sci. Int'l., 33, 83-88. Ritchie JM and Greene NM (1990) Local anesthetics. Chapter 15. In The Pharmacological Basis of Therapeutics (A. Oilman and L. S. Goodman, (eds.)) Pergamon Press, New York, p 311. Ritz MC, Lamb RJ, Goldberg SR and Kuhar MJ (1987) Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 237, 1219-1223. Ruth TJ and Wolf AP (1979) Absolute cross sections for the production of 18F via the 18O(p,n)i8F reaction. Radiochimica. Act, 26, 21-24. Scheffel U, Pogun S, Stathis M, Boja JW and Kuhar MJ (1991) In vivo labeling of cocaine binding sites on dopamine transporters with [3H]WIN 35,428. /. Pharm. Exp. Ther., 251, 954-958. Schwartz WJ, Sharp FR, Gunn RH and Evarts EV (1976) Lesions of ascending dopaminertic pathways decrease forebrain glucose uptake. Nature, 251, 155-157. Shiue C-Y, Fowler JS, Wolf AP, Watanabe M and Arnett CD (1985) Synthesis and specific activity determinations of no-carrier-added (NCA)18F-labeled butyrophenone neuroleptics - benperidol, haloperidol, spiroperidol and pipamperone. J. Nucl. Med., 26, 181-186. Sihver W, Nordberg A, Langstrom B, Mukhin AG, Keren AO, Kimes AS and London ED (2000) Development of ligands for in vivo imaging of cerebral nicotinic receptors. Behavioral Brain Researchll3, 143-157. Simpson GM, Shih JC, Chen K, Flowers C, Kumazawa T and Spring B (1999) Schizophrenia, monoamine oxidase and cigarette smoking. Neuropsychopharmacology, 20, 392-394. Singer T (1995) Monoamine oxidases: old friends hold many surprises. FASEB J. ,9, 605-610. Spealman RD, Kelleher RT and Goldberg SR (1983) Stereoselective effects of cocaine and a phenyltropane analog. J. Pharm. Exp. Ther. ,225, 509-514. Stein EA, Pankiewicz J, Harsch HH, Cho J-K, Fuller S, Hopffmann RG, Hawkins M, Rao SM, Bandetti PA, and Bloom AS (1998) Nicotine-induced cortical activation in the human brain: a functional MRI study. Am. J. Psych., 155, 1009-1015. Telang FW, Volkow ND, Levy A, Logan J, Fowler JS, Felder C, Wong C and Wang G-J (1999) Distribution of tracer levels of cocaine in the human brain as assessed with averaged [HC]cocaine images. Synapse,31, 290-296. Volkow ND, Fowler JS, Wolf AP, Schlyer D, Shiue C-Y, Dewey SL, Alpert R, Logan J, Christman D, Bendriem B, Hitzemann R and Henn F (1990) Effects of chronic cocaine abuse on postsynaptic dopamine receptors. Am. J. Psych., 147, 719-724. Volkow ND, Fowler JS, Wolf AP, Hitzemann R, Dewey SL, Bendriem B, Alpert R and Hoff A (1991) Changes in brain glucose metabolism in cocaine dependence and withdrawal. Am. J. Psych., 148, 621-626.

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Volkow ND, Hitzemann R, Wang G-J, Fowler JS, Wolf AP and Dewey SL (1992a) Long-term frontal brain metabolic changes in cocaine abusers. Synapse, 11, 184-190. Volkow ND, Fowler JS, Wolf AP, Wang G-J, Logan J, MacGregor R, Dewey SL, Schlyer D and Hitzemann R. (1992b) Distribution and kinetics of HC-cocaine in the human body measured with PET. J. Nucl. Med., 33, 521-525. Volkow ND, Fowler JS, Wang G-J, Hitzemann R, Logan J, Schlyer DJ, Dewey S, and Wolf AP (1993) Decreased dopamine D2 receptor availability is associated with reduced frontal metabolism in cocaine abusers. Synapse, 14, 169-177. Volkow ND, Wang G-J, Fowler JS, Logan J, Schlyer D, Hitzemann R, Lieberman J, Angrist B, Pappas N, MacGregor R, Burr G, Cooper T and Wolf AP (1994) Imaging endogenous dopamine competition with [ U C] raclopride in the human brain. Synapse, 16, 255-262. Volkow ND, Ding Y-S, Fowler JS, Wang G-J, Logan J, Galley SJ, Dewey S, Ashby C, Liebertman J and Wolf AP (1995) Is methylphenidate like cocaine? Studies on their pharmacokinetics and distribution in human brain. Arc. Gen. Psych., 152, 456-463. Volkow ND, Wang G-J, and Fowler JS, Galley SJ, Ding Y-S, Logan J, Hitzemann R and Lieberman J (1996) Relationship between psychostimulant induced high and dopamine transporter occupancy. Proceedings Nat' I Acad. Set USA, 93, 10388-10392. Volkow ND, Wang, G-J, Fischman M, Foltin R, Fowler JS, Abumrad NN, Vilkun S, Logan J, Galley SJ, Pappas N, Hiizemann R and Shea CE (1997a) Relationship between subjective effecls of cocaine and dopamine transporter occupancy. Nature, 386, 827-830. Volkow ND, Wang G-J, Fowler JS, Logan J, Galley SJ, Hiizemann R, Chen AD, Dewey SL and Pappas N (1997b) Decreased slrialal dopaminergic responsiveness in detoxified cocaine-dependent subjects. Nature, 386, 830-833. Volkow ND, Wang G-J, Fowler JS, Galley SJ, Logan J, Ding Y-S, Hiizemann R and Pappas N (1998) Dopamine iransporter occupancies in ihe human brain induced by therapeutic doses of oral methylphenidate. Am. 7. Psych., 155, 1325-1331. Volkow ND, Wang G-J, Fowler JS, Logan J, Galley SJ, Wong C, Hiizemann RJ and Pappas N (1999a) Reinforcing effecls of psychoslimulanls in humans are associated wilh increases in brain dopamine and occupancy of D2 receptors. J. Pharm.Exp. Ther., 291, 409-415. Volkow ND, Wang GJ, Fowler JS, Logan J, Galley SJ, Gifford A, Hiizemann R, Ding Y-S and Pappas N (1999b) Prediction of reinforcing responses to psychoslimulanls in humans by brain dopamine D2. Am. J. Psych., 156, 1440-1443. Volkow ND and Fowler JS (2000) Addiction, a disease of compulsion and drive: Involvemeni of ihe orbilopfronlal cortex. Cerebral Cortex 10, 318-325. Volkow ND, Wang G-J, Fowler JS, Logan J, Gerasimov M, Maynard L, Ding Y-S, Galley SJ, Gifford A and Franceschi D (2001) Therapeutic doses of oral meihylphenidale significanlly increase exlracellular dopamine in ihe human brain. J. Neurosci., 21 (RC121 ),l-5.

20. RESEARCH AND CLINICAL APPLICATION OF NEURORECEPTOR IMAGING HENRY N. WAGNER, JR. AND ZSOLT SZABO Johns Hopkins Medical Institutions, Baltimore, MD, USA.

INTRODUCTION In 1966 Al Wolf came to Johns Hopkins for a National Institutes of Health site visit to review our proposal to have the NIH fund a project based on obtaining a cyclotron to produce positron emitting radionuclides. This was to be part of our existing program-project grant entitled NUCLEAR INSTRUMENTATION AND CHEMISTRY IN MEDICINE. This grant is now in its 40th year of continual operation, having been peerreviewed every five years. At the time, Al was well known as a radiochemist who had begun working in radiation chemistry during the Manhattan Project that resulted in the development of the atomic bomb. Our site visit was his first exposure to the field that has come to be known as Nuclear Medicine. He was so attracted by the promise of the field that he teamed up with his Brookhaven National Laboratory colleagues to direct his existing radiochemistry program in biomedical directions. Paradoxically, our request for supplementary funds to install a cyclotron was not granted, because we had also requested funds to create a new site for the cyclotron. At that time, the United States government funded the establishment of 20 new medical schools, on the mistaken assumption that if there were more doctors the costs of medicine would decrease because of competition. We were not able to incorporate a cyclotron into our program until 1979, when we altered our existing space to make room for a cyclotron. It was then funded as part of a neurosciences oriented proposal that was approved by the National Institutes of Health. In the intervening years it had become clear that cyclotron-produced F-18 fluorodeoxyglucose could be used to reveal the regions of the brain that were activated by mental functions. Our research was to be directed to the design, production, validation and application of positron emission tomography to the study of the chemistry of neurotransmission in experimental animals and the living human brain. The existence of chemical "receptors" in the brain was first proposed over a century ago by Claude Bernard who postulated their existence because chemicals such as curare could have such profound mental effects, far beyond what he believed could be produced by a direct effect. In 1905 Langley extended the concept of neuroreceptors, and a few years later Paul Ehrlich emphasized the important role that receptors could play in pharmacology (Langley, 1905; Ehrlich, 1909; Schuster, 1962). Ehrlich emphasized the concept that drugs were "magic bullets" that interacted with chemicals naturally present in the body. He pointed out that our ability to smell exceedingly small quantities of specific molecules was an example of the process. At present, the greatest application of the use of radioligands that bind to receptors has been in the design, development and application of drugs. Since the first receptor imaging in a living human being by positron emission Handbook of Rudiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wilev & Sons, Ltd

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tomography (PET), much more has been learned about how drugs affect the brain than about how the brain works. Radiotracer technology has taught us a great deal about which regions of the brain are involved in sensory activation, movement, and other mental functions, and helps select those regions in which the chemistry of neurotransmission can be examined. One can identify those regions of the brain that contain specific neuroreceptors and quantify their availability to radioligands that bind to these specific receptors. Such knowledge can form the means to develop drugs intended to stimulate or inhibit the neuronal pathways related to mental functions. Today we take for granted that chemicals within our brains affect our mental activity, but as recently as 50 years ago, neurophyiologists believed that communication from one neuron to another was entirely electrical, and hoped that measurement of the electrical activity of the brain would make it possible to relate brain activity to mental functions. They could not imagine that chemical processes, which they believed could not possibly take place so rapidly, could determine the complexity and extent of neuronal activity in the brain. We now know that they can. Not only is there co-valent bonding, but also the "bumping, fitting and sticking" of complicated stereospecific simple and complex molecules with one another occurs in inconceivable numbers and speed. Characterizing and quantifying the regional distribution of these radioligand/receptor interactions has only recently become possible because the receptors are present in extremely low concentrations, amounting to approximately 10 to 100 pmol/gram of brain, which amounts to about one millionth of the weight of the brain of a human being. In addition to having adequate sensitivity, the radioligands must be sufficiently specific since they can also be bound to proteins, lipids and carbohydrates in a non-specific fashion not related to information transfer. Often the amount of the radioligand bound to the specific receptor under study is low compared to the non-specific binding, thereby creating "noise" in the system. This is less of a problem in in vitro measurements, but is especially troublesome in studies of receptors in the brains of experimental animals or living persons. To differentiate non-specific from specific binding, one can saturate the specific sites, which usually have a higher affinity, and then measure the non-specific binding, subtracting the results from the measurements of the total binding of the radioligand to both specific and non-specific binding sites. Such experiments depend on the development of radioligands with high specific radioactivity (that is, the amount of radioactivity per millimol of ligand) so that the amount bound to the specific receptors is much higher than that bound to the non-specific binding sites, which only bind the radioligand after the specific sites have been saturated.

EFFECTS OF DRUGS ON THE BRAIN The earliest work to lead to the recognition of the importance of receptors was based on the study of insulin and estrogen receptors. This was followed by research on neuroreceptors, highlighted by the studies in 1966 by Hornykiewicz, who found that patients with Parkinson's disease had diminished amounts of dopamine in the striatum secondary to degeneration of the neurons in the substantia nigra (Hornykiewicz 1966). The

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nigro-striatal pathway from the substantia nigra to the caudate nucleus and putamen accounts for over 70% of brain dopamine. The administration of L-DOPA, a precursor of the neurotransmitter dopamine, was helpful in the care of patients with Parkinson's disease, a "dopamine-deficiency disease." Over thirty years ago it was found that drugs which bound to dopamine receptors activated the enzyme, adenylate cyclase, which catalyzed the reaction of ATP becoming cyclic AMP. This reaction was only weakly stimulated by other catecholamines, such as norepinephrine and isoproterenol. Drugs affecting mental functions are of two types: stimulating (agonist) agents; and inhibiting (antagonist) agents. Dopamine antagonists that bind to dopamine receptors on post-synaptic neurons diminish delusions and hallucinations in psychotic patients, with the unfortunate side effect of producing Parkinsonian-type symptoms. Other examples of antagonist (inhibitory) drugs include cimetidine, which binds to histamine receptors and propanolol, which binds to beta-adrenergic receptors. The drug valium stimulates benzodiazepine receptors, which in turn brings about an inhibitory effect on neuronal activity.

IDENTIFICATION OF RECEPTORS In 1971 Goldstein et al., observed that the morphine analogue levophanol bound stereospecifically to subceliular fractions of the mouse brain (Goldstein et al., 1971). In 1975 Creese et al. found specific binding of tritiated dopamine and haloperidol to receptors in extracts of the brain of experimental animals (Creese et al., 1975). One year later, they reported that the degree of inhibition of the binding of the radioligands to the receptors by different therapeutic drugs in patients with schizophrenia was related to the potency of their ability to block the binding of the radioligands to the dopamine receptors (Creese et al., 1976). For the first time, a pharmacological effect could be related to a binding reaction to receptors in the brain. These in vitro binding studies could provide a basis for initial screening of putative therapeutic Pharmaceuticals. In 1978 Kuhar et al. used autoradiography to image the regional concentrations of dopamine receptors in the basal ganglia of rats (Kuhar et al., 1978). Their results led to attempts to extend analogous studies of neuroreceptors to living experimental animals and human beings by the measurement of radioligand binding in vivo, using the newly developed technique of positron emission tomography (PET).

POSITRON EMISSION TOMOGRAPHY (PET) In 1977, Sokoloff and colleagues at the National Institutes of Health in Bethesda, Maryland, used the radiotracer, carbon-14 deoxyglucose (DG), an analogue of glucose, to delineate functionally activated regions of the brain (Sokoloff et al., 1977). Because carbon-14 emits only beta particles, the tracer distribution after intravenous injection in living rats could only be examined by autoradiography. Nevertheless, they were able to show that increased brain function, such as vision, increased accumulation of the C-14 DG. The tracer is transported into the brain and then binds to the enzyme hexokinase, which phosphorylates the deoxyglucose. The metabolism then stops after phosphorylation. The tracer remains in

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situ, its amount of binding reflecting the level of regional neuronal activity. Concurrent with SokolofPs studies, the technique of positron emission tomography was being developed by Ter-Pogossian, Phelps and Hoffman at Washington University in St. Louis, and by Yamamoto, Robertson and others at Brookhaven National Laboratory in New York (Robertson et al., 1973; Ter-Pogossian et al., 1975). Unlike the beta-emitter, carbon-14 DG, fluorine-18 fluorodeoxyglucose (FDG) emits positrons, which then immediately interact with electrons with subsequent emission of high energy photons (511 kev) which leave the point of interaction (the process is called annihilation). Each of the photons produced by interaction leave in opposite directions. This directionality permits accurate determination of the location of the F-18 atoms within the brain by means of positron emission tomography. The FDG is injected intravenously during a control period or during mental stimulation, such as vision, hearing, or movement of a part of the body, and between 30 and 60 minutes later serial images are made of the distribution of the FDG in volume elements throughout the brain. These images reveal the sites of neuronal activation by depicting increased rates of accumulation of the tracer in the various regions of the brain. The spatial resolution with present day studies is approximately 3 cubic mm. With appropriate mathematical models and computer software, one can create 3-dimensional "functional images" reflecting regional hexokinase and related mental activity. FDG accumulation is increased in regions of the brain known to be related to vision (visual cortex), hearing (auditory cortex), motion or other mental activity whenever the subject is stimulated by vision, sound or movement. THE DOPAMINERGIC SYSTEM DOPAMINE RECEPTORS Initially, in vitro autoradiography after the administration of receptor-binding ligands, and subsequently in vivo positron emission tomography, has improved our knowledge of the location and characteristics of dopamine receptors and fostered studies of the effects of age, and pathological conditions such as Parkinson's disease, schizophrenia and other neuropsychiatric disorders. The first successful imaging of a neuroreceptor in a living human was carried out in 1983 (Wagner et al., 1984), using the radiolabelled neuroleptic drug, carbon-11 N-methyl spiperone (C-ll NMSP). NMSP is an analogue of the drug, spiperone, an antagonist of the D2 dopamine receptor used with some success to treat patients with schizophrenia. Its administration resulted in a decrease in hallucinations and delusions. Using serial imaging of the regional concentrations of the C-ll NMSP, it is possible to quantify the regional distribution of the receptors in units of picomoles per gram. In general, radiolabelled ligands for the study of neuroreceptors fall into two categories: (a) irreversibly bound ligands, such as NMSP, and (b) reversibly bound ligands with lower receptor affinity, such as C-ll raclopride. Kinetic modeling makes it possible to use measurements of the rate of entry of the tracer into the regions of interest containing the receptors and compare them to those regions of the brain that do not contain receptors. In the case of radioligands with low affinity that are reversibly bound, equilibrium can be achieved

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after a short period of time subsequent to the intravenous injection. A mathematical model is used to calculate receptor density. The details of the method for quantification of regional receptor availability is described elsewhere (Wong et al., 1997). Soon after the first successful imaging of dopamine receptors in living human by PET, studies were carried out in normal subjects and patients with Parkinson's disease and schizophrenia. Since the expression of several neuroreceptors differs among different species, the ability to carry out such studies in humans in the healthy and diseased states was a great step forward. There are also differences in receptor status in postmortem specimens compared to living subjects. Also, many persons whose brain tissue becomes the subject of post-mortem examination may have received drugs that altered neuroreceptors during their lifetime. This is particularly true for patients with Parkinson's disease or schizophrenia. Finally, in most cases there are no good experimental animal models of neuropsychiatric disease. For al! these reasons, the studies of living subjects offer enormous advantages. Prior to the PET studies, it was well known that binding to specific neuroreceptors on post-synaptic neurons was involved in therapeutic effects on specific neuronal populations, including the dopaminergic system. It was also known that neurological diseases were associated with loss of specific neuronal types. Assessment of the availability of specific receptors promised to provide biological markers for the intactness of specific neuronal populations and provide objective criteria for diagnosis and the planning and monitoring of treatment, particularly pharmacological treatment. The ability to differentiate neuronal loss from functional loss of the binding sites remains a challenge. It is well accepted that certain psychiatric and some neurological diseases are not due to neuronal cell loss, but are related to molecular dysfunction. Neurons known to secrete dopamine as a neurotransmitter lie in the substantia nigra (so-called because of it high concentrations of the pigment melanin) and extend axons to the caudate and putamen (the basal ganglia) along the so-called nigro-striatal pathway, and into the limbic cortex, which includes the amygdaloid nucleus, hippocampus, anteromedial frontal cortex, and the medial and lateral habenula (the mesolimbic pathway). There are five types of dopamine receptors. The different types are identified by the binding characteristics of different ligands and therapeutic effects. The five known types of dopamine receptors are grossly divided into two classes: the Dl-like receptors and the D2-like receptors. Dl and D5 receptors are assigned to the first group while D2, D3 and D4 receptors are assigned to the second. Further division of the dopamine receptor types is complex and rather confusing. Many of the neurons that secrete dopamine as a neurotransmitter have an inhibitory effect. At times increased dopaminergic activity results in a positive behavioral effect because it removes the inhibitory effect of dopaminergic neurons. Apomorphine stimulates dopamine receptors (an "agonist" effect) and reduces the firing of other neurons. Behavior is the result of the summed interactions of many neurotransmitters and receptors that integrate stimulatory and inhibitory effects. It can be said that it is a lot easier to study the effects of drugs on receptors and correlate them with desired functional effects than it is to "find out how the brain works."

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In Parkinson's disease, many of the most disturbing symptoms involve movement. Post-mortem studies indicated that dopamine concentrations in the caudate nucleus and putamen (basal ganglia) were reduced in patients with Parkinson's disease. In fact, this finding is what led to the important discovery that the administration of L-DOPA, the precursor of dopamine, was effective in alleviating the symptoms of the disease. Drugs such as bromocryptine bind to dopamine receptors and have a stimulatory effect on dopaminergic neurons, which is associated with an improvement in symptoms. The first significant observation of dopamine receptor imaging studies with PET was that there was a striking decrease in available dopamine receptors in the caudate nucleus and putamen in normal persons between the ages of 19 and 73, with most of the decrease occurring before the age of 40 (Wong et al., 1984). Because the radioligand used was not affected by endogenous dopamine, it was thought to reflect the loss of post-synaptic dopaminergic neurons, rather than a reduction of dopamine function.

THE DOPAMINE TRANSPORTER In 1971, Axelrod described the reuptake of norepinephrine from the synapse into the pre-synaptic nerve terminals (Axelrod, 1971). Pre-synaptic uptake sites for monoamines, including dopamine, decrease synaptic concentrations, and are believed to be responsible for inactivation of the effects of the neurotransmitters, in this case, dopamine. The therapeutic potency of anti-depressant drugs increases in proportion to their degree of binding to the monoamine reuptake sites. Little is known of the role of reuptake sites in physiology, but the effects of psychoactive drugs have been studied extensively. The reuptake of synaptic dopamine into pre-synaptic neurons has been described by Uhl and Johnson (Uhl & Johnson, 1991). The dopamine transporter was first cloned in 1991 by Kilty et al and Shimada et al. (1992). The dopamine transporter (DAT), a NaVCl -dependent membrane protein (Giros & Caron, 1993; Nelson 1998) has the function to recycle synaptic dopamine (DA) and regulate its synaptic concentration. The monoaminergic transporters DAT, NET (norepinephrine transporter) and SERT (serotonin transporter) possess 12 transmembrane domains that are responsible for binding of cocaine and MPP*. The function of DAT is modulated by the pre-synaptic DA autoreceptors, which can affect both the release and the reuptake of the neurotransmitter (Senders et al., 1997). Within the brain the DAT is expressed only by the dopaminergic neurons and is much less widespread than the DA receptors. The distribution of DAT parallels that of DA innervation (Ciliax et al., 1995; Freed et al., 1995) with high densities in the striatum and nucleus accumbens and less density in the globus pallidus, cingulate cortex, olfactory tubercle and amygdala. In addition to dopaminergic axon terminals, DAT is also detected on the perikarya, dendrites and axons of midbrain DA neurons. The psychostimulant effects of cocaine and amphetamine (Ritz et al., 1987) are due to increased synaptic dopamine caused by antagonism of DAT (cocaine) or by enhanced release of DA (amphetamines). In an effort to decrease craving for cocaine and other abused drugs, several drugs have been developed that block the uptake of dopamine by the dopamine transporter.

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DAT has been implicated as a candidate gene in Parkinson's disease, schizophrenia, Tourette's syndrome, attention-deficit disorder, and substance abuse. Polymorphisms of the DAT have been observed in attentiondeficit hyperactivity disorder (ADHD) and some forms of Parkinson's disease (Le Couteur et al,, 1997). SCHIZOPHRENIA An increased functioning of the dopaminergic system has been postulated in schizophrenia. This hypothesis is supported by postmortem autoradiography and ligand-binding studies, which demonstrate increased density of D2-like receptors in schizophrenic brain tissues (Seeman, 1992; Seeman et al., 1987). In vivo PET studies with the high affinity ligand ["CJmethylspiperone (Gjedde et al., 1996; Nordstrom et al., 1996; Nordstrom et al., 1995; Wong et al., 1986) have confirmed these observations while PET studies performed with the low affinity ligand [nC]raclopride (Farde et al., 1988) have not. The discrepancy has in part been explained by the dimerization of the D2 receptors and a difference in the binding affinities to the monomeric and dimeric forms (Zawarynski et al., 1997; Ng et al., 1996; Lee et al, 1997). The increased binding of ["Cjmethylspiperone may be related to an increased presence of the receptor monomers. Since the total density of the receptors is unchanged, [nC]raclopride, which binds both to the monomer and the dimer, demonstrates no change (Nordstrom et al., 1995). The D4 receptor has also been proposed as a candidate receptor for schizophrenia. Using post-mortem schizophrenic brains and [3H]NGD 94-1, an increase in dopamine D4 receptors has been found in the entorhinal cortex and the substantia nigra (Seeman et al., 1993). Other interesting hypotheses postulate abnormal coupling between the Dl and D2 receptors or altered synaptic concentrations in the striatum of schizophrenic patients (Seeman et al., 1989; Seeman et al., 1993). Although the expression and function of the DA receptors are not yet completely clarified, PET measurements have been successfully used for design and testing of psychopharmaceutical drugs. For example, PET studies have revealed important relationships between the occupancy of D2 receptors in neuroleptic-treated patients and the improvement of the clinical symptoms. Specifically, they revealed that the antipsychotic effect was achieved with a receptor occupancy rate of 70-80%, while extrapyramidal side effects were observed with 80% occupancy (Briicke et al., 1992a; Farde et al., 1988; Farde et al., 1992a; Farde et al, 1992b; Pilowsky et al, 1992). PARKINSON'S DISEASE A combination of multiple mechanisms is probably responsible for the development of idiopathic Parkinson's disease (PD). The four main concepts include oxidative damage, environmental toxins, genetic predisposition, and accelerated aging. The concepts involving the effects of endogenous toxicity (accumulation of oxygen radicals) and exogenous toxicity (incorporation of environmental toxins similar to MPTP) are long-standing. More recent studies have concentrated on genetic mechanisms. One of these studies focused on autosomal recessive juvenile parkinsonism (AR-JP), one of the most common forms of familial parkinsonism. PARK-2, a gene responsible for AR-JP was recently identified in Japan along with its putative protein product called parkin (Kitada et al., 1998). A wide variety of different mutations of PARK-2

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have been observed in European patients with AR-JP (Abbas et al., 1999). In 2000, researchers at Duke University Medical Center demonstrated that alteration of the parkin gene contributes to both the common late-onset form of Parkinson's disease, and the rarer, early-onset form of the disease (http://wwwchg.mc.duke.edu/Parkin%20Gene.html). Alterations of two enzymes (UCH-L1 and a-synuclein) have been linked to an autosomal dominant form of PD (Polymeropoulos et al., 1997; Leroy et al., 1998). These enzymes are associated with the ubiquitin cascade of protein degradation. Parkin probably also participates in the same cascade and is identical or analogous to the enzyme ubiquitin-protein ligase. Mutant parkins show loss of the ubiquitin-protein ligase activity. The resulting accumulation of proteins mat have yet to be identified causes neural cell death. Abnormal protein ubiquitination has been implicated not only in Parkinson's disease but also in other neurodegenerative diseases characterized by involvement of abnormal proteins, such as Alzheimer disease and amyotrophic lateral sclerosis (Shimura et al., 2000). The histopathological hallmark of PD is loss of DA neurons in the midbrain substantia nigra with consequential dopaminergic denervation of the basal ganglia. This is followed by reduction of the chemical and molecular markers of DA innervation such as the DAT. Typically, reduction of DAT is more pronounced in the putamen (75 %) than in the caudate (64 %) or nucleus accumbens (Miller et al., 1997).

CO2CH3

WIN-35,428

Figure 1: Chemical structure of the dopamine transporter (DAT) radioligand WIN-35,428 and PET images obtained 55-75 minutes post injection in a patient with Parkinson's disease (PD) and an age matched healthy subject. In this patient with a relatively mild PD, Hoehn-Yahr stage 2, there is marked reduction in the density of DAT in the putamen and relatively preserved density in the head of the caudate. A range of cocaine derivatives have been synthesized for PET and SPECT imaging of the DAT and have been used to demonstrate reductions of DAT even in mild PD (Frost et al., 1993; Innis et al., 1993; Guttman et al., 1997; Tissingh et al., 1998; Fischman et al., 1998). Potential clinical applications are manifold: a) differential diagnosis of parkinsonism, b) early detection of PD, c) assessment of disease severity, and d)

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follow-up of tissue transplantation therapy. There is a decrease in regional dopamine transporters with age, and an accelerated decrease in patients with Parkinson's disease (Figure 1) (Frost et al., 1993; Kuikka et al,, 1997). Studies with !8F-CFT and PET show that there is a progressive decrease in dopamine transporter as the symptoms of Parkinson's disease progress (Nurmi et al., 2000). LESCH-NYHAN DISEASE AND GILLES DE LA TOURETTE'S SYNDROME Lesch-Nyhan is an X-linked genetic disease in children caused by loss of hypoxanthine-guanine phosphoribosyl transferase. PET imaging demonstrates a 50-75 % reduction of striatal DAT density (Wong et al., 1996; Ernst et al., 1996). Behavioral changes are severe and can be modulated by modifications of DA function. The mechanism of Gilles de la Tourette's syndrome is unknown. The role of the DA system is implicated by the fact that therapeutic manipulations reducing DA activity are beneficial for alleviation of the symptoms. Imaging studies indicate an increase in striatal DAT density (Malison et al., 1995). THE SEROTONERGIC SYSTEM Serotonin acts both as a neurotransmitter and a neuromodulator and is involved in the regulation of mood, appetite, sleep, sexual desire, cognition and memory. The bodies of the serotonergic neurons reside in the brainstem raphe nuclei, and in the reticular formation and pons. Their axons project to virtually every region of the brain. The highest densities of axon terminals are found in structures related to the limbic system such as the hypothalamus, the amygdala and the corpus striatum. Although functionally very important, somewhat less dense serotonergic innervation is found in the cingulate gyrus and cerebral cortex and the density of these nerve terminals is lowest in the cerebellum. Serotonin, like the monoamines dopamine and norepinephrine as well as the diamine histamine, is found in axonal varicosities from which it can be released in a nonjunctional fashion. Two important molecular mechanisms are involved in serotonin release, the serotonin autoreceptors and the serotonin transporter. The larger portion of serotonin is probably released nonsynaptically, which underscores the role of serotonin in sustained brain functions (Bach-y-Rita, 1993). The first two families of 5-HT receptors (5-HT1 and 5-HT2) were discovered as a result of radioligand binding studies and the distinct properties of the dopaminergic radioligand [3H]spiperone to bind to these receptors with variable affinities. The list of identified receptors grew very rapidly in part due to an increasing number of autoradiography or ligand binding studies and more recently due to the development of cloning techniques and functional assays. There are now seven known 5-HT receptor families: 5-HT1 (subtypes 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5-HT1P, 5-HT1S), 5-HT2 (subtypes 5-HT2A, 5HT2B, 5-HT2C), 5-HT3, 5-HT4, 5-HT5 (5-HT5A, 5-HT5B), 5-HT6, and 5-HT7 (Baez et al., 1995; Murphy et al, \ 998). Members of each of these receptor families have similar molecular biological, pharmacological and biochemical properties. They differ from each other in their relative affinities for serotonin and in their speed of response. Members of the 5-HT1 receptor family as well as the receptors 5-HT6 and 5-HT7 are functionally coupled to the GTP-protein (G-protein coupled receptors) and have a relatively slow response.

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The 5-HT3 receptor is a fast acting ion channel receptor. The diversity in ligand affinity and cellular signal transduction mechanisms explains the wide range of roles of serotonin in the CNS (Schofield et al., 1990). Subtle differences in the primary amino acid sequences in the receptor protein result in dramatic differences in ligand selectivity. Additionally, it has recently been discovered that drugs such as atypical antipsychotic drugs and serotonin-selective reuptake inhibitors may interact with a large number of heretofore unknown 5HT receptors. Clozapine, for instance, has high affinity for at least four separate 5-HT receptors, and it is unknown which of these receptors is essential for its unique therapeutic efficacy (Kroeze & Roth , 1998). Another important component of the serotonergic system is the serotonin transporter (SERT), a Na+, Cldependent plasma membrane transporter (Hoffman et al., 1998). The primary mechanism for termination of monoaminergic neurotransmission is through reuptake of released neurotransmitter by the SERT. Immunohistochemistry demonstrates that the plasma membrane transporters are present along axons, soma, and dendrites despite of the fact that its mRNA is concentrated in the raphe nuclei and in the dorsomedial nucleus. SERT mRNA is dramatically downregulated during immobilization stress of the experimental animal (Hoffman et al, 1998). Molecular imaging with PET and SPECT represents a more recently introduced direct technique with many advantages for investigation of the serotonin system: 1) It provides regional information about individual brain structures 2) It is noninvasive as it does not require biopsy of brain tissue 3) It provides quantitative information which can be used for between subject and within subject comparisons 4) It permits investigation of multiple components such as the monoamino oxydase enzyme, the serotonin receptors and the SERT

5-HT1A RECEPTOR 5-HT1A receptors participate in the control of sexual behavior, appetite, thermoregulation, sleep and cardiovascular function and are involved in the pathogenesis of obsessive-compulsive disorder, impulsivity and alcoholism. The main therapeutic potential of 5-HT1A receptors has been in the treatment of anxiety and depression, since 5-HT1A ligands with agonist activity possess antianxiety, antidepressant, antiaggressive, and perhaps anticraving, anticataleptic, antiemetic, and neuroprotective properties. One of the radioligands which has been introduced for quantitative studies of 5-HT1A receptors is [!1C]WAY-100635. Its binding potential (BP) correlates with the know distribution of these receptors and is particularly high in cerebral cortex, hippocampus and raphe nuclei, whereas low BP is observed in the basal ganglia and thalamus (Ito et al., 1999). [HC]WAY-100635 has been used to study the pharmacologically selectivity and potency of 5-HT1 receptor specific drugs (Andree et al., 1999).

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5-HT2A RECEPTOR 5-HT2A receptors play a role in appetite control, thermoregulation, sleep, cardiovascular function and muscle contraction. Several 5-HT2A antagonist are currently in clinical trials as potential antipsychotic agents as they often bind to the dopamine receptors as well. Certain types of schizophrenia may actually be more responsive to the combined effect than to the effect of selective dopamine antagonists. 5-HT2A receptors are also involved in the actions of classical hallucinogens. At first, the nonselective radioligand, [HC]N-methylspiperone was used for imaging of both the dopamine D2 receptors of the striatum (Wagner et al., 1983; Wagner et al., 1984) and serotonin 5-HT2 receptors of the cortex (Mayberg et al, 1988). Later, [18F]altanserin was introduced as a more specific 5-HT2A radioligand (Lemaire et al., 1991; van Dyck et al, 2000). 5-HT TRANSPORTER (SEROTONIN TRANSPORTER, SERT) The serotonin transporter (SERT) represents an exclusive and quite stable constituent of the serotonergic neuron. While the number of serotonin receptor types and subtypes is large, there is only one SERT. SERT is expressed by the serotonergic neurons which is the main advantage compared to the serotonin receptors which can reside both on pre-synaptic (serotonergic) and post-synaptic (nonserotonergic) neurons. Loss of serotonin terminals due to neurodegenerative processes or the action of neurotoxins results in reduced density of the SERT, an important relationship that represents the rationale for utilization of PET and SPECT to image the SERT. Various radioligands have been tested as potential candidates for imaging the SERT. Desirable attributes of such a radioligand are high selectivity and affinity for the SERT, high stability in vitro, high stability in physiologic solutions and favorable lipophilicity. The first successful clinically applicable radioligand, [ U C] McN5652 ((1,2,3,5,6,10p-hexahydro-6[4-(methylthio) phenyl] pyrrolo[2,l-a]isoquinoline), fulfills these requirements at least in part and permits examination of the SERT using PET. This ligand demonstrates a high cerebral uptake and a hypothalamus/cerebellar ratio of 6.2 in rodents. Images of the human brain obtained with the pharmacologically active enantiomer [nC](+)McN56562 are of high quality (Figure 2) (Szabo et al, 1995; 1996; 1999). Another potent SERT ligand, 5-iodo-6-nitro-2-piperazinylquinoline, has been labelled with iodine-123 and used as a radioligand for SPECT imaging. This ligand has also been labeled with bromine-76 for PET imaging (Lundqvist et al., 1999). Beta-CIT, a tracer of the dopamine transporter (DAT) has also been used for SPECT imaging of the SERT of the midbrain (Tauscher et al., 1999). Treatment with fluoxetine resulted in a 40% reduction of binding in the midbrain, confirming specific binding of this radioligand to the SERT.

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Figure 2: Sagittal section of PET scans of the brain of a healthy human subject obtained 55-95 minutes after injection of [11C](+)McN5652 (active enantiomer) and [11C](-)McN5652 (inactive enantiomer) in comparison with the coregjsteied MRI image. There is high specific binding in the midbrain and pons. PET Imaging is well suited for detection of subclinical monoamine depletions in humans (McCann & Ricaurte, 1993) and has been used to demonstrate reduction of SERT in MDMA treated animals (Scheffel & Ricaurte, 1990; Scheffel et al, 1996; Scheffel et al., 1998) and in humans users of MDMA (McCann et al., 1998; Semple et al., 1999). 5-HT2A receptors have also been studied in MDMA users with SPECT and [123I]-5-I-R91150 as a radioligand (Reneman et al., 2000). These studies demonstrated reduced binding to the SERT, increased binding to the serotonin receptors and correlation of these findings with impaired cognitive function in MDMA users. PARKINSON'S DISEASE Parkinson's disease (PD) is a neurodegenerative condition that primarily affects the dopamine system, but the loss of serotoninergic and norepinephrinergic neurons has also been described. It is often complicated by depression and the prevalence rates of depression in this patient group have been reported to be as high as 40%. Selective serotonin reuptake inhibitors (SSRIs) are being considered as first-line therapy for PD patients with depression (Gimenez-Roldan et al., 1996; Cummings & Masterman, 1999). The successful application of SSRIs implicates the role of serotonin in depression of PD and motivates studies of the serotonin system of PD in addition to dopamine. PET imaging studies reveal reduced binding to the SERT in Parkinson's disease (Szabo et al., 1996). These observations correlate with the observation that the short allele of 5-HTTLPR occurs more often in patients with Parkinson's disease who have high scores on tests of depression and anxiety (Ricketts et al., 1998; Menza et al., 1999). Iodine-123-beta-CIT (2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane) has been used for SPECT imaging of both DAT and SERT. Striatal binding of the radioligand correlated with Hoehn-Yahr stage and motor

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function (UPDRS). Hypothalamic/midbrain binding which reflects the SERT did not significantly correlate with either Hoehn-Yahr stage or UPDRS scores including both motor and nonmotor measures (Kim et al., 1999), ALZHEIMER'S DISEASE Allelic functional variations of serotonin transporter expression also represent a susceptibility factor for late onset Alzheimer's disease (Li et al., 1997). Altered serotonin platelet uptake (Kumar et al., 1995) and altered SERT binding in the raphe and hippocampus (Tejani-Butt et al., 1995) have been described in Alzheimer's disease. In addition to disorders of mood there is also increasing evidence that progressive loss of serotonergic function contributes to deficiencies of the cholinergic system and results in diminishing cognitive function. Imaging studies will play an increasing role in the assessment of the serotonin system of aged individuals, particularly in those with Alzheimer's disease (Meltzer et al., 1998). PET imaging studies with [!8F]altanserin have shown reduced binding to the 5-TH2A receptors in the anterior cingulate, prefrontal cortex, and sensorirnotor cortex of patients with Alzheimer's disease (Meltzer et al., 1999). DEPRESSION Scores of evidence emphasize the role of the serotonin system in depression (Mann, 1999): 1) SSRIs are the most successful drugs to treat depression 2) Dietary manipulation of the serotonin precursor amino acid L-tryptophan affects mood 3) Serotonin releasing drugs result in blunted response in depressed subjects 4) Some studies demonstrated reduced CSF levels of the serotonin metabolite 5hydroxyindoleacetic acid (5-HIAA) in patients with depression 5) Proteins related to the serotonin system such as the SERT, 5-HT2 receptor or MAO-B are altered in depressed patients 6) Autoradiographic studies on brain specimens of suicide victims demonstrated altered serotonin receptors, for example increased 5-HT2 receptor binding sites (Agren et al., 1991) 7) Altered signal transduction in the cerebral cortex and altered regulation of serotonin neurons in the dorsal raphe in depression and suicide (Stockmeier, 1997) The effect of the serotonin releasing drug, D-fenflurarnine (which has been removed from the US market for its toxic effects) has been measured using [18FJFDG/PET. In depressed patients brain activation was reduced compared to controls, particularly in the frontal area (Mann et al., 1996a). These findings were confirmed by measurements of the 5-HT receptors in the frontal cortex and [!8F]altanserin uptake was found reduced in a region of the right hemisphere including the postero-lateral orbito-frontal cortex and the anterior insular cortex with a trend to similar changes in the left hemisphere (Biver et al, 1997). In vivo PET imaging with [!IC]WAY-100635 has been used to measure radioligand binding to the 5-HT1A receptor in depression. The mean 5HT1A receptor binding potential was reduced in the raphe and in the mediotemporal cortex in the depressed subjects relative to controls. Post hoc comparisons showed that reduction in 5-HT1A receptor binding was not limited to these regions, but extended to the occipital cortex and postcentral gyrus as well. The magnitude of these abnormalities was most prominent in bipolar

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depressives and unipolar depressives with bipolar relatives (Drevets et al., 1999). Reduced 5-HT1A receptor binding was not changed by selective serotonin reuptake inhibitor treatment (Sargent et al., 2000). The SERT has been imaged in subjects with major depression by quantifying the binding of the nonselective SPECT radioligand [!23I]p-CIT in the brainstem (Malison et al., 1998), thalamus and hypothalamus (Willeit et al., 2000). Determination of the allelic variation of the SERT in patients with major depression with psychotic features demonstrated that homozygotes for the long variant (1/1) of the SERT promoter and heterozygotes (1/s) showed a better response to fluvoxamine than homozygotes for the short variant (s/s). In the group treated with fluvoxamine plus pindolol all the genotypes acted like 1/1 treated with fluvoxamine alone. Fluvoxamine efficacy in delusional depression seems to be related to allelic variation within the promoter of the SERT gene. Even though other factors may be implicated, genotyping of the SERT promoter combined with PET or SPECT imaging of the SERT represents a promising tool to individualize the pharmacological treatment of depression (Smeraldi et al., 1998). Following desipramine treatment, depressed patients show a significant decrease in 5-HT2 receptor binding of [18F]setoperone in frontal, temporal, parietal, and occipital cortical regions. A pre-treatment decrease in 5HT2 receptor binding was observed bilaterally and was particularly prominent in the frontal cortex (Yatham et a/.,1999). The mean frontal to cerebellum radioactivity concentration ratio, an index of the fl8F]setoperone specific binding to 5-HT2A receptors, was higher in treated than in untreated patients when age was taken into account. This suggests that chronic treatment by SSRIs could induce an up-regulation of the 5-HT2A receptors, and that 5-HT2A receptor down-regulation is not a common mechanism for the therapeutic effects of all serotoninergic antidepressive drugs (Massou et al., 1997). Measurements of the 5-HT2A receptor in neuroleptic-naive patients with schizophrenia demonstrated reduced binding in the frontal cortex supporting previously reported postmortem studies (Ngan et al., 2000). Reduced cortical binding of [nC]N-methylspiperone (NMSP) has also been seen in drug-free but previously treated schizophrenic patients (Okubo et al., 2000), suggesting that cortical 5-HT2 receptors may be altered by therapy. PET imaging proved very useful for assessment of the potency of antipsychotics and assessment of the optimal therapeutic dose to minimize side effects. For example, PET has been used to demonstrate that conventional neuroleptics induce high D2 receptor occupancy (70-90 %) and that extrapyramidal side effects increased at D2-receptor occupancies above 80%. Standard doses of clozapine induced low (20-67%) D2receptor occupancy, but very high (85-90%) 5-HT2A receptor occupancy (Farde et al, 1995). The novel antipsychotics risperidone and olanzapine induced high occupancy of both D2 and 5-HT2A receptors at suggested standard doses (Nyberg et al., 1998; 1999). 5-HT2A occupancy rates are associated with favorable treatment for depressive symptoms of schizophrenia and improvement of cognitive function (Kasper et al., 1999).

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SUMMARY The great advances in radiopharmacology and PET technology in the recent years contributed significantly to better understanding of the role of the dysfunction of serotonin and dopamine receptors and transporters in normal brain and diseases such as Parkinson's disease or schizophrenia. These advances also contributed to development and testing of new psychopharmaceuticals for treatment of neurological and psychiatric disorders. ACKNOWLEDGMENT We appreciate the editorial comments of Mrs. Julia Buchanan and the support by the NIH (grant numbers: DA 5707, DA 6275, AG 14400, and AA 11653). REFERENCES Abbas N, Lucking CB, Ricard S, Durr A, Bonifati V, De Michele G, Bouley A, Vaughan JR, Gasser T, Marconi R, Broussolle E, Brefel-Courbon C, Harhangi BC, Oostra BA, Fabrizio E, Bohme GA, Pradier L, Wood NW, Filla A, Meco G, Denefle P, Agid Y and Brice A (1999) A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. French Parkinson's Disease Genetics Study Group and the European Consortium on Genetic Susceptibility in Parkinson's Disease. Human Molecular Genetics 8:567-574. Agren H, Reibring L, Hartvig P, Tedroff J, Bjurling P, Hornfeldt K, Andersson Y, Lundqvist H and Langstrom B (1991) Low brain uptake of L-[llC]5-hydroxytryptophan in major depression: a positron emission tomography study on patients and healthy volunteers. Acta. Psyciatr. Scand. 83:449-455. Andree B, Thorberg SO, Halldin C and Farde L (1999) Pindolol binding to 5-HT1A receptors in the human brain confirmed with positron emission tomography. Psychopharmacology (Berlin) 144:303-305. Axelrod J (1971) Noradrenaline: Fate and control of its biosynthesis. In Les Prix Nobel en 1970, Stockholm: Imprimerical Royal PA Norstedt and Soner, pp. 189-208. Bach-y-Rita P (1993) Nonsynaptic diffusion neurotransmission (NDN) in the brain.. Neurochem Int 23:297318. Baez M, Kursar JD, Helton LA, Wainscott DB and Nelson DL (1995) Molecular biology of serotonin receptors. Obes. Res. 3 Suppl 4:441S-447S. Biver F, Wikler D, Lotstra F, Damhaut P, Goldman S and Mendlewicz J (1997) Serotonin 5-HT2 receptor imaging in major depression: focal changes in orbito-insular cortex. Br. J. Psychiatry 171:444-448. Briicke T, Roth J, Podreka I, Strobl R, Wenger S and Asenbaum S.(1992) Striatal dopamine D2-receptor blockade by typical and atypical neuroleptics. Lancet 1992;339:497. Ciliax BJ, Heilman C, Demchyshyn LL, Pristupa ZB, Ince E, Hersch SM, Niznik HB and Levey Al (1995) The dopamine transporter: immunochemical characterization and localization in brain. J. Neurosc. 15:1714-1723. Creese I, Burt DR and Snyder SH (1975) Dopamine receptor binding: differentiation of agonist and antagonist states with 3H-dopamine and 3H-haloperidol. Life Sci. 17:933-1001. Creese I, Burt DR and Synder S (1976) Dopamine receptor binding predicts clinical and pharmacological potencies of anti-schizophrenic drugs. Science 192: 481-483.

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Cummings JL and Masterman DL (1999) Depression in patients with Parkinson's disease. Int. J. Geriatr. Psychiatry 14:711 -718. Drevets WC, Frank E, Price JC, Kupfer DJ, Holt D, Greer PJ, Huang Y, Gautier C and Mathis C (1999) PET imaging of serotonin 1A receptor binding in depression. Biol. Psychiatry 46:1375-1387. Ehrlich P (1909) Uber den jetzigen Stand der Chemotherapie. Ber. Dtsch. Chem. Ges. 42:17-47. Ernst M, Zametkin AJ, Matochik JA, Pascualvaca D, Jons PH, Hardy K, Hankerson JG, Doudet DJ and Cohen RM (1996) Presynaptic dopaminergic deficits in Lesch-Nyhan disease. N. Engl. J. Med. 334:1568-1572. Farde L, Wiesel F-A, Halldin C and Sedvall G (1988) Central D2-dopamine receptor occupancy in schizophrenic patients treated with antipsychotic drugs. Arch Gen. Psychiatry 45:71-76. Farde L (1992a) Selective Dl- and D2-dopamine receptor blockade both induces akathisia in humans—a PET study with [UC]SCH 23390 and ["CJraclopride. Psyche-pharmacology 107:23-29. Farde L, Nordstrom A-L, Wiesel F-A, Pauli S, Halldin C and Sedvall G (1992b) Positron emission tomographic analysis of central Dl and D2 dopamine receptor occupancy in patients treated with classical neuroleptics and clozapine. Arch Gen. Psychiatry 49:538-544. Farde L, Nyberg S, Oxenstierna G, Nakashima Y, Halldin C and Ericsson B (1995) Positron emission tomography studies on D2 and 5-HT2 receptor binding in risperidone-treated schizophrenic patients. J. Clin. Psychopharmacol 15:19S-23S. Fischman AJ, Bonab AA, Babich JW, Prather Palmer E, Alpert NM, Elmaleh DR, Callahan RJ, Barrow SA, Graham W, Meltzer PC, Hanson RN and Madras BK (1998) Rapid detection of Parkinson's disease by SPECT with altropane: a selective ligand for dopamine transporters. Synapse 29:128-141. Freed C, Revay R, Vaughan RA, Kriek E, Grant S, Uhl GR and Kuhar MJ (1995) Dopamine transporter immunoreactivity in rat brain. J. Comp. Neurol. 359:340-349. Frost JJ, Rosier AJR, Reich SG, Smith JS, Ehlers MD, Snyder SH, Ravert HT and Dannals RF (1993) Positron emission tomographic imaging of the dopamine transporter with "C-WIN 35,428 reveals marked declines in mild Parkinson's disease. Ann. Neurol. 34:423-431. Gimenez-Roldan S, Dobato JL, Mateo D, Gonzalez Alvarez M, Novillo Infantes MJ and Gimenez-Zuccarelli M (1996) Depression and Parkinson's disease: neurobiologic foundations and therapeutic management Neurologic 11:332-340. Giros B and Caron MG (1993) Molecular characterization of the dopamine transporter. Trends Pharmacol Sci. 14:43-49. Gjedde A, Reith J and Wong DF (1996) In schizophrenia, some D2-like receptors are still elevated. Psychiatry Res. 67:159-162. Goldstein A, Lowney LI and Pal BK (1971) Stereospecific and nonspecific interactions of the morphine congener levorphanol in subcellular fractions of mouse brain. Proc. Natl. Acad. Sci. 68:1742-1747. Guttman M, Burkholder J, Kish SJ, Hussey D, Wilson A, DaSilva J and Houle S (1997) ["C]RTI-32 PET studies of the dopamine transporter in early dopa-naive Parkinson's disease: implications for the symptomatic threshold. Neurol. 48:1578-1583. Hoffman BJ, Hansson SR, Mezey E and Palkovits M (1998) Localization and dynamic regulation of biogenic amine transporters in the mammalian central nervous system. Front Neuroendocrinol. 19:187-231. Hornykiewicz O (1966) Dopamine (3-hydroxytyramine) and brain function. Pharmacol. Rev. 18:925-64.

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21. DYNAMIC NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET WYNNE K. SCHIFFERn AND STEPHEN L. DEWEYn f

NYU School of Medicine, Department of Psychiatry, New York, NY 10016, USA. ^Brookhaven National Laboratory, Chemistry Department, Upton NY 11973, USA.

INTRODUCTION Positron emission tomography (PET) has become a valuable interdisciplinary tool for understanding physiological, biochemical and pharmacological functions at a molecular level in living humans, whether in a healthy or diseased state. The utility of tracing chemical activity through the body transcends the fields of cardiology, oncology, neurology and psychiatry. In this, PET techniques span radiochemistry and radiopharmaceutical development to instrumentation, image analysis, anatomy and modeling. PET has made substantial contributions in each of these fields by providing a venue for mapping dynamic functions of healthy and unhealthy human anatomy. As diverse as the disciplines it bridges, PET has provided insight into an equally significant variety of psychiatric disorders. Using the unique quantitative ability of PET, researchers are now better able to noninvasively characterize normally occurring neurotransmitter interactions in the brain. With the knowledge that these interactions provide the fundamental basis for brain response, many investigators have recently focused their efforts on an examination of the communication between these chemicals in both healthy volunteers and individuals suffering from diseases classically defined as neurotransmitter specific in nature. In addition, PET can measure the biochemical dynamics of acute and sustained drug abuse. Thus, PET studies of neurotransmitter interactions enable investigators to describe a multitude of specific functional interactions in the human brain. This information can then be applied to understanding side effects that occur in response to acute and chronic drug therapy, and to designing new drugs that target multiple systems as opposed to single receptor types. Knowledge derived from PET studies can be applied to drug discovery, research and development (for review, see (fowler et al., 1999; Burns et al., 1999)). Here, we will cover the most substantial contributions of PET to understanding biologically distinct neurochemical systems that interact to produce a variety of behaviors and disorders. Neurotransmitters are neither static nor isolated in their distribution. In fact, it is through interactions with other neurochemically distinct systems that the central nervous system (CNS) performs its vital role in sustaining life. Exclusive quantitative capabilities intrinsic to PET make this technology a suitable experimental tool to measure not only the regional distribution of specific receptors and their subtypes, but also the dynamic properties of neuroreceptors and their inherent influence on related neurotransmitter pathways. The ability to investigate

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dynamic properties in a non-invasive and reproducible manner provides a powerful tool that can extend our current knowledge of these interactions.

Coupled with innovative paradigms including pharmacologic

manipulations, physiologic models and reconstruction theories, knowledge derived from PET studies can greatly advance our understanding of normal and abnormal brain function. TECHNICAL ATTRIBUTES SPECIFIC TO NEUROTRANSMITTER MAPPING WITH PET Neurotransmitter mapping with PET is based on the principle that labeled compounds can be designed to compete with endogenous neurochemical activity at receptor sites on both pre- and post-synaptic terminals. In this, radiotracers can be targeted at a number of physiological functions, from pre-synaptic release and reuptake to post-synaptic uptake. In the midst of disease states, this information provides vital insight into the development of potential pharmacologic interventions (Ding et al., 1995; Ginovart et al., 1997; Frey et al., 19%; Meyer et al., 1999). Further, neurochemical mapping with PET can be carefully controlled, so that depending on the kinetic properties of the tracer molecule, varying degrees of competition interfere with normal neurotransmitter activity and occupy all receptor sites, or less competitively occupy only a few of the receptor sites.

Several concepts are fundamental to understanding the general foundation of mapping

dynamic neurochemical interactions with PET. These are the choice of a ligand and target neurotransmitter system, kinetic modeling of chosen radioligands, and specific to multi-neurotransmitter investigations, the design of a challenge paradigm. From this, we can assay the effects of Pharmaceuticals on interrelated neurotransmitter systems as well as gather information about regular and irregular endogenous states. THE LIGAND The freedom implied by the ability to label a variety of compounds might lead one to raise the possibility of labeling the neurotransmitters themselves, but this is impossible for several reasons. Primarily, neurotransmitters do not cross the blood-brain barrier. Because labeled compounds must be injected or inhaled, blood-brain barrier permeability is necessary. Second, the binding affinity of all amino acid and amine neurotransmitters to their specific receptors is low. In this, the fate of neurotransmitters themselves cannot be controlled (i.e., presynaptic reuptake or enzymatic degredation), but the action of a molecule designed to bind to a specific receptor, or ligand, can be reliably predicted depending on the kinetics of the labeled molecule. Therefore, it is necessary to use molecules that bind to the receptor under investigation with a known affinity. The choice of a ligand should be clinically relevant, and its actions should be well documented. In this, ligands should have a known affinity for specific receptor subtypes to minimize the possibility of confounding effects or untraceable radioligand activity from promiscuous ligand delivery. Second, ligands should have a biological half-life long enough to remain in systemic circulation for the duration of the scanning period (although kinetic modeling parameters can compensate for this). Finally, it is important that ligands have a conformation that can be readily labeled while retaining blood-brain barrier permeability. By using a competitive ligand with a high affinity, the amount of neurotransmitter bound to receptor sites will be insignificant compared with the amount of ligand bound to identical sites. Theoretically, noncompetitive ligands with moderate affinity do not interfere with endogenous neurotransmitter activity

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(Seeman et al., 1990). Investigations that use competitive ligands identify fundamental neuroreceptor arrangement, and typically differentiate basal receptor physiology in healthy and unhealthy psychiatric conditions. For example, the high affinity D2 receptor radiotracer "C-N-methylspiroperidol (1!C-NMSP) demonstrated a two- to three-fold increase in ligand competition in schizophrenic patients when compared to normal controls (Wong et al., 1986), while the noncompetitive D2 radiotracer 11C-raclopride demonstrated no change in competition between schizophrenic patients and controls (Farde et al., 1987). A later discovery was that endogenous dopamine (DA) released into the synapse competes with 11C-raclopride for D2 receptor binding sites (Seeman et al., 1990), whereas 11C-NMSP is less sensitive to the influence of endogenous dopamine (Logan et al., 1992) and possibly a more effective tool for measuring receptor physiology. Further, while Farde et al., demonstrated no change in the binding characteristics of 11C-rac3opride between schizophrenic patients and normal controls, Breier et al., recently demonstrated amphetamine-induced increases in dopamine release produce less 11C-raclopride binding in schizophrenic patients when compared to healthy controls given the same challenge (Breier et al., 1997). Together, these studies support the existence of similar endogenous dopamine activity between schizophrenic patients and controls, while demonstrating substantially different responses to a challenge of the dopamine system. In this, measurement of dynamic neurotransmitter activity may be more informative about the plasticity of neurochemical activity in the brain than about a single physiological trait like receptor concentration. KINETIC MODELING The scientific progression from investigations of static neuroreceptor concentration to dynamic neurotransmitter activity also instigated marked innovations in the methods by which we process and statistically manipulate data from PET cameras. Specifically, the initial receptor mapping studies by Wong (Wong et al., 1986) and Farde (Farde et al., 1987) used the same radioactive label on two compounds with different pharmacokinetic properties. 11C-raclopride, used by Farde et al., reaches equilibrium during the time of the scan and allows the quantification of binding parameters via a Scatchard plot. 11C-NMSP, however, does not reach equilibrium within the period of the scan and requires kinetic modeling (Tune et al., 1993). It is now possible to estimate changes in endogenous dopamine following a pharmacologic challenge in each PET study with a mathematical approach (for example, see (Dewey et al., 1992a)). Since extracting absolute measures of neurotransmitter concentrations requires knowledge of both receptor concentration (B max ) and the in vivo receptor-ligand dissociation constant, kd, and estimations of Bmax are crude and difficult to measure (Logan et al., 1997), results from most dynamic PET studies are expressed as relative change between baseline (radiotracer alone) and challenge scans (i.e., pretreatment + challenge drug + radiotracer). Studies in our laboratory were designed to examine changes in receptor availability by determining parameters that are linearly related to free receptors, rather than the actual number of free receptors targeted by conventional neuroreceptor investigations. For noncompetitive, moderate affinity radiotracers, two such modeling parameters are the distribution volume (DV) and Bmax/Kd. The distribution volume is a graphical method of analysis applied to ligands that bind reversibly to receptors or enzymes. A method for determining DV was developed for the kinetic analysis of 11C-cocaine data and has been applied to the analysis of 11C-benztropine and 11C-raclopride data used in our investigations of neurotransmitter activity (Logan et al., 1990;Dewey et al., 1990c).

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Alternatively, the DV can also be calculated from the kinetic parameters determined from a non-linear least squares fit of the data using a compartmental model. For a three-compartment model, the steady state DV (KR) is given by K 1 /k 2 (l+k 3 /k 4 ). For a two-compartment model, the ratio KR is expressed by K]/k2- For the analysis of 11C-cocaine data, kinetic parameters determined using the graphical technique are consistent with estimates from a nonlinear least squares method. In addition, the ratio Bmax/Kd is similar to that found in vitro (Logan et al., 1990). One of the advantages of applying the DV approach is that it represents a linear function of free receptor concentration and does not depend on blood flow, since the variables containing blood flow cancel in the ratio Ki/k2- Specifically, if we calculate the ratio of the DV for a region containing few or no target receptors to the DV for a receptor-rich region, the K 1 /k 2 ratio can be eliminated, giving a parameter more closely related t o Bmax/Kd. A s a validation o f t h e D V ratio approach, Koeppe e t a/., (Koeppe e t al., in ligand delivery (K1) to the visual cortex. In this investigation, the DV for 11C-flumazenil in the visual cortex or other regions was unaltered, supporting the insensitivity of the DV parameter to focal alterations in blood flow. The DV parameter is also easily computed and less sensitive to noise than the individual kinetic parameters, which often have large standard errors associated with their determination. This technique is conceptually similar to the two-compartment model approach used by Frey et al., (Frey et al., 1990) to study muscarinic receptors with ''C-tropanyl benzilate and by Koeppe et al., (Koeppe et al., 1991) to study benzodiazepine receptors with 11C-flumazenil. In sum, our investigations estimate the free concentration of tracer in tissue and the concentration of nonspecifically bound tracer from a reference region of the brain that contains few or no target receptors. For 11C-raclopride and 11C-benztropine, the cerebellum provides a reference region containing few or no dopamine or cholinergic terminals or receptors, so ligands for these receptors distribute only to the compartments of free ligand and nonspecifically bound ligand. Data from PET studies reported here is expressed as the percentage change relative to the baseline DV ratio of radiotracer binding. NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET Neurodevelopmental models support the involvement of large numbers of discrete neurons involved in adaptive plasticity, and contend that these processes underlie psychopathology (Olney & Farber, 1995). It is also likely, however, that many diverse neurotransmitter systems contribute to maintain a certain level of homeostasis. Whether a healthy or unhealthy state, there remains a dynamic interplay between endogenous systems to sustain the organism. Noncompetitive receptor ligands have been established as successful markers of endogenous neurotransmitter activity, and are sensitive to pharmacologic challenges that either increase or decrease synaptic concentrations of the target neurotransmitter. Our laboratory and others have taken this paradigm one step further to explore the functional interactions between isolated neurotransmitter systems. To do this, we measured the acute response of one neurotransmitter system to a challenge targeting a discrete, related system. Prior to evaluating the feasibility of PET as an accurate measure of simultaneous neurotransmitter interactions, it was essential to select for study a well-defined neurotransmitter system whose function is

DYNAMIC NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET

607

dependent on interactions between at least two chemically different neurotransmitter systems. Furthermore, the target system should be particularly sensitive to alterations in either of these endogenous neurotransmitters and their site of interaction must be localized to neurochemical factors that are within the resolution of the machine itself. Within the confines of these requirements, the extra-pyramidal motor system provides a multi-neurotransmitter region within the window of PET resolution. Interconnections of the extra-pyramidal motor system have been extensively documented in animal and human postmortem studies using a wide variety of neuroanatomical, neurophysiological and behavioral methods. The principle components of this system involve interactions between acetylcholine, dopamine, gamma aminobutyric acid (GABA) and serotonin. Disruptions in any single component of this system present a discrete clinical picture. For example, therapeutically relevant blockade of dopamine D2 receptors produces movement disorders, which can be alleviated by decreasing cholinergic activity. Studies using a variety of methods have demonstrated that these neurochemical systems interact in the corpus striatum through reciprocal inhibitory and excitatory connections with the substantia nigra and medial raphe nucleus (Arnfred & Randrup, 1968; Bloom et al., 1965; Costall & Olley, 1971; Ehlert et al., 1981; Lehmann & Langer, 1982). The corpus striatum, consisting of the caudate nucleus and putamen, is well within the resolution of modern PET scanners and contains a large number of dopamine D2 and cholinergic muscarinic receptors. While each of these neurotransmitters is involved in maintaining the necessary input required for the normal day-to-day operation of the extra-pyramidal motor system, each has been implicated in other physiological roles that may or may not be directly linked to the neuronal systems mentioned above. These other roles may contribute to disease states of the CNS and/or conditions that predispose or maintain drug addiction. MODULATION OF DOPAMINERGIC ACTIVITY STUDIED WITH 11C-RACLOPRIDE AND 18FNMSP The interpretation of neurotransmitter interaction studies is dependent on the ability of each radiotracer to demonstrate receptor specific binding and reproducibility, and also on the ability of each radiotracer to reflect fluctuations in receptor availability secondary to drug-induced changes in endogenous neurotransmitter activity. Therefore, in a series of PET studies, we utilized a multi-mechanistic approach to investigate the effects of increases in endogenous dopamine on the binding of 11C-raclopride. Using d-amphetamine and GBR-12909, drugs that alter synaptic dopamine concentrations by different mechanisms, we addressed the question of whether 11C-raclopride binding is sensitive to changes in endogenous dopamine. In the first set of studies, we used 11-amphetamine to increase synaptic dopamine levels by releasing cytostolic stores into the synapse. Following d-amphetamine administration, the ratio of striatal to cerebellar DV for 11Craclopride decreased by an average of 16%. Parallel studies using in vivo microdialysis techniques in freely moving rats demonstrate af-amphetamine significantly increases extracellular striatal DA release. GBR12909, a potent dopamine reuptake inhibitor, decreased the same ratio by an average of 22%. Again, this investigation was confirmed by microdialysis studies indicating an increase in extracellular dopamine release following GBR-12909 pretreatment (Dewey et al., 1993b). Together, these studies demonstrated that our system was capable of measuring changes in synaptic dopamine concentrations, and prompted the utility of

608

HANDBOOK OF RADIOPHARMACEUTICALS

this strategy to quantify changes in dopamine activity secondary to pharmacologic modulation of related, upstream neurotransmitter systems. For a summary of each investigation and the result, please see Table 1.

Table 1. Effects of neurotransmitter-specific pharmacological challenges on dopaminergic activity measured with PET Neurotransmitter System

Radioligand

Drug Challenge

Challenge Effect

Radioligand Response

Dopamine Activity

Dopamine

" C-raclopride

cocaine, amphetamine

•ODA

£

ft

benztropine, scopolamine

ft ACh

£

ft

I8

Acetylcholine

Serotonin

GABA

F-NMSP

"C-raclopride

altanserin citalopram

$ 5-HT

fts-m

-0ft

ft -0-

11

vigabatrin lorazepam

tf GABA ttGABA

ft

-0-

C-raclopride

GABA/ dopamine

11

C-raclopride

GVG + cocaine

ft GABA, ft DA

No change

No change

Glutamate

11

C-raclopride

Phencyclidine

tfGlu

$

ft

C-raclopride

GVG + phencyclidine

ft GABA, DGlu

No change

No change

Glutamate/ dopamine

11

CHOLINERGIC MODULATION OF DOPAMINE Initially, our investigations of dynamic neurochemical interactions measured with PET looked at the cholinergic/dopaminergic system, with the outcome measure being changes in endogenous dopamine activity. The cholinergic system has demonstrated an essential role in many memory and cognitive functions (Davies & Verth, 1977; Meltzer & Stahl, 1976). Some studies have hypothesized that cholinergic hyperactivity may underlie the negative symptoms associated with schizophrenic illnesses as well (Tandon & Greden, 1989). The highest levels of choline acetyltransferase and cholinergic receptors in the human CNS are found in the striatum and the majority of cholinergic cells are interneurons (Cortes et al., 1987; Fibiger, 1982). These cholinergic interneurons provide excitatory input to local GABA neurons, which in turn are thought to inhibit the activity of dopaminergic neurons (Bunney & Aghajanian, 1976).

DYNAMIC NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET

609

Our initial primate investigations demonstrated the binding of I8F-NMSP was reduced by 13% after the animals received an injection of benztropine (an anticholinergic agent) when compared to control animals receiving no benztropine (Dewey et al., 1990a). This result was confirmed in human subjects where the uptake of 18F-NMSP in the striatum was reduced by an average of 10% after benztropine pretreatment (Dewey et al., 1988)1. In a related study, we used 11C-raclopride as the radiotracer and scopolamme as the cholinergic challenge drug, as it has a more selective mechanism of action than benztropine. Human subjects treated with scopolamine demonstrated a decrease in striatal 11C-raclopride binding of 17%, indicating greater increases in endogenous dopamine competition after scopolaniine when compared to benztropine (Dewey et al., 1993c). In sum, decreased brain acetylcholine levels secondary to benztropine or scopolamine treatment appear to increase brain dopamine, indicated by decreased I8F-NMSP binding. These data are consistent with the aforementioned model of a multisynaptic feedback loop linking cholinergic neurons indirectly to dopaminergic neurons via GABAergic interneurons (Bunney & Aghajanian, 1976). By decreasing cholinergic activity, benztropine inhibits the excitatory input to GABAergic neurons, reducing GABAergic inhibition of dopaminergic neurons. Subsequent disinhibition of dopaminergic neurons produces a higher level of synaptic dopamine release, and increased competition with 11C-raclopride at postsynaptic D2 receptors. These studies demonstrate that in some circumstances, acetylcholine regulates brain dopamine release. SEROTONERGIC MODULATION OF DOPAMINE Further studies investigating the modulation of dopaminergic systems focused on the possible modulation of dopamine by serotonin, as interactions between these systems have been well documented. Functionally, serotonin has been implicated in sleep, aggression, pain transmission, Alzheimer's disease, normal aging, affective disorders and suicide (Wesemann et al., 1983; Stanley & Mann, 1984; Pucilowski & Kostowski, 1983; Bowen et al., 1983; Crow et al., 1984; Middlemiss et al., 1986; Reynolds et al., 1984). In addition, the greater affinity of several atypical neuroleptics, non-benzodiazepine anxiolitics and new antidepressant agents (e.g., selective serotonin reuptake inhibitors, SSRIs) for cortical serotonin receptors has been linked to their therapeutic efficacy. Electrophysiological, biochemical and behavioral evidence indicates ascending serotonergic pathways from the medial and dorsal raphe modulate the function of mesolimbic and mesostriatal dopamine systems (Joyce et al., 1993; Joyce, 1993; Zazpe et al., 1994; Kapur & Remington, 1996). Our initial primate studies used the 5-HT? receptor antagonist altanserin as a pretreatment drug found that 1!C-raclopride binding was reduced by 37% in the striatum (Dewey et al., 1995), indicating diminished 5-HT activity actually stimulated dopamine release. In the same study, increasing 5-HT activity with citalopram (SSRI) decreased synaptic dopamine levels and increased striatal 11C-raclopride binding. These studies were later confirmed by Tsukada et al., who decreased 5-HT with ketanserine (similar to altanserin) and found decreased "C-raclopride binding in the striatum, consistent with increased dopamine release (Tsukada etal., 1999).

610

HANDBOOK OF RADIOPHARMACEUTICALS

By decreasing serotonergic activity with altanserin, we quantified secondary increases in brain dopamine by measuring decreases in 11C-raclopride binding (indicating increased competition from endogenous dopamine). Next, we increased serotonergic activity with citalopram administration and demonstrated decreases in endogenous dopamine release with increased 11C-raclopride binding. In an attempt to extend this finding to human subjects, we used fenfluramine, a selective 5-HT reuptake inhibitor and releasing agent, to increase serotonergic activity (Smith et al., 1997). Our results, contrary to our previous findings in baboons, indicated that increasing 5-HT activity increased DA activity, as evidenced by a 13% decrease in 11C-raclopride binding. These results are consistent with more recent studies indicating chronic (Tiihonen et al., 1996) or acute (Vollenweider et al., 1999) increases in serotonergic transmission produced increases in synaptic dopamine concentrations, as evidenced by PET and "C-raclopride binding. The difference between our primate and human results may be due to the broad serotonergic effect of fenfluramine versus altanserin, the effects of anesthesia on the baboon, or species-specific effects. Increasing serotonergic activity with fenfluramine produced subsequent increases in serotonergic activity measured by decreased "C-raclopride binding. GABAERGIC MODULATION OF DOPAMINE We have also used the pharmacological challenge paradigm with PET to study the GABA/dopamine interaction. The finding that GABA functions as the most common inhibitory neurotransmitter in the CNS resulted in its implication, either directly or indirectly, in the pathogenesis of several neurodegenerative conditions. These conditions include Huntington's and Parkinson's disease, epilepsy, schizophrenia and tardive dyskinesia. In addition, studies have shown that GABAergic activity modulates central neurotransmitter systems targeted by drugs of addiction (DeFeudis, 1984; Koob, 2000; Dewey et al., 1998). Thus, numerous PET studies have been aimed at the study of GABAergic systems and their contributions to disease and addictive states. There are several inherent difficulties in perturbing the GABA receptor complex, including the controversy over the GABA receptor subtypes and their functional role in the striatum. The availability of specific GABA receptor agonists or antagonists that reliably affect central GABA levels without producing profound disruptions in CNS function has also been a concern. The administration of gamma-vinyl GABA (GVG, vigabatrin), a suicide inhibitor of the GABA catabolizing enzyme GABA transaminase, has been used to successfully enhance GABAergic neurotransmission through increased brain GABA levels. This compound is used clinically to treat epilepsy, and it has been shown to reliably elevate endogenous GABA levels after systemic administration without affecting other amino acid transmitters. According to the multisynaptic feedback loop described, we would expect an inhibitory influence of GABA on the activity of dopaminergic neurons and hence an increase in "C-raclopride binding. To test this, "Craclopride in baboons was imaged prior to and following the administration of GVG (Dewey et al., 1992a). The result was an average increase of 25% in "C-raclopride binding, indicating significantly depressed synaptic DA activity. In the second part of the study, lorazepam, a clinically prescribed benzodiazepine administered intravenously prior to the scan, led to an average increase of 22% in striatal "C-raclopride

DYNAMIC NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET

611

binding. This PET study provided evidence that dopaminergic neurons of the substantia nigra and the ventral tegmental area are responsive to pharmacological alterations in GABA activity. Interestingly, a similar investigation by Hietala et al., (Hietala et al., 1997) found weeklong pretreatment with lorazepam had no effect on striatal

1!

C-raclopride binding in human patients, but acute lorazepam increased

11

C-raclopride

binding in primates. In sum, increasing whole brain GABA levels with vigabatrin produced subsequent decreases in dopamine release, as measured by increases in 11C-raclopride binding. GABAergic MODULATION OF DOPAMINE IN SUBSTANCE ABUSE We have also used 11C-raclopride binding and PET to explore the inhibitory potential of GVG on the brain response to substances of abuse. The rewarding effects of psychostimulants have been associated with their ability to increase striatal dopamine levels (Volkow et al., 1999). The system of primary interest in these conditions involves the mesocorticolimbic dopaminergic projections from the ventral tegmental area (VTA) and substantia nigra into the ventral striatum, medial prefrontal cortex, and amygdala, which participate in the neural processing of motivated behavior (Robbins et al., 1989). Alterations in this system are thus hypothesized to play roles in mediating the euphoria and behavioral reinforcement associated with drugs of abuse (for review, see (McBride et al., 1999)). Specifically, the nucleus accumbens (NAcc) has demonstrated the greatest sensitivity to changes induced by drugs that inhibit dopamine reuptake, stimulate dopamine release, or increase dopamine through neurotransmitter system interactions. This evidence is derived from microdialysis studies in rats showing higher extracellular fluid dopamine concentrations after administration of psychostimulants (Di Chiara et al., 1993; Di Chiara et al., 1992).2 In our extensive PET studies utilizing the strategy of increasing GABAergic activity with GVG as a mechanism for preventing psychostimulant induced dopamine release, we correlate striatal "C-raclopride binding in primates with microdialysis investigations of NAcc dopamine release in rodents. This allows us to better relate the neural responses from various drug challenges with the many available rodent models of substance abuse (craving, reward and reinforcement), as well as assess the temporal window for pretreatment strategies. Our initial studies in the substance abuse arena demonstrated that GVG restores striatal 1! C-raclopride binding to normal levels following acute cocaine administration in baboons (Dewey et al., 1997). In other words, while a cocaine challenge diminished 11C-raclopride binding, pretreatment with GVG before cocaine produced a degree of 11C-raclopride binding undistinguishable from animals given 11C-raclopride alone. In further support of this strategy, GVG effectively modulated basal reward thresholds produced by cocaine administration (Kushner et al., 1997), abolished the conditioned place preference produced by cocaine (Dewey et al., 1998), and reduced self-administration of cocaine (Kushner et al., 1999) and heroin (Xi & Stein, 2000) in rodents. Additionally, we have demonstrated that increasing GABAergic activity with GVG can modulate decreases in 11C-raclopride binding produced by nicotine and heroin (Dewey et al., 1999; Gerasimov et al., 1999), consistent with microdialysis studies (Gerasimov et al., 1999). To further investigate the specific properties of GVG that contribute to its efficacy as a potential antiaddictive agent, as well as to justify the ability of 11C-raclopride to detect changes induced by multiple

612

HANDBOOK OF RADIOPHARMACEUTICALS

pharmacologic manipulations, we administered the pure enantiomers of the racemic compound before an acute nicotine challenge (Schiffer et al., 2000). Our previous investigations demonstrating increases in striatal dopamine concentrations after nicotine administration served as the basis for this investigation. Where nicotine diminished 11C-raclopride binding by ~12%, pretreatment with (R,S)-GVG and active S(+)GVG increased 11C-raclopride binding 9 and 8%, respectively. Figure 1 presents the radioactivity distribution of "C-raclopride (red). Here, it is clear that nicotine diminishes D2 receptor occupancy (a), in that there is visibly diminished 11C-raclopride binding. Pretreatment with S(+)-GVG prevents nicotineinduced dopamine release (b) and restores receptor occupancy to near test/retest values, demonstrated by increased 11C-raclopride binding. Additionally, pretreatment with the inactive R(-)-GVG decreased 11Craclopride binding after a nicotine challenge by ~13%. These results support studies indicating that, as an anticonvulsant, the active S(+) enantiomer also retains the pharmacologic efficacy of GVG. Our microdialysis data corroborate our PET studies in that S(+)-GVG was as effective as the racemic compound at decreasing the extracellular NAcc dopamine response to cocaine in rodents. Figure 1. Radioactivity distribution of 11C-raclopride (a) after nicotine alone (0.3mg/kg) and (b) after S(+)-GVG pretreatment (150 mg/kg) prior to the same nicotine challenge.

GABAergic MODULATION OF DOPAMINE IN SCHIZOPHRENIA The clinical potential of enhanced GABA transmission described above is most likely attributable to decreases in dopaminergic activity, believed to be a common denominator in many schizophrenic psychoses (Wassef et al., 1999). The effects of enhanced GABA function on dopaminergic pathways in the human brain are of particular interest for studies on biological factors in schizophrenia. These studies have formulated a GABA hypofunction hypothesis of schizophrenia, which postulates a deficient GABAergic inhibition in schizophrenia and subsequent alterations in other neurotransmitter systems, including DA hyperactivity in the brain (Keveme, 1999; Egan & Weinberger, 1997; Weinberger, 1997). To further explore this latter hypothesis, we administered the NMDA antagonist phencyclidine (PCP) to primates and measured 11C-raclopride binding with PCP alone and in the presence of increased GABAergic

613

DYNAMIC NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET

activity with GVG. The NMDA antagonist model of schizophrenia has surpassed previous dopaminergic theories in both clinical and basic science investigations (Javitt & Zukin, 1991). Administration of NMDA antagonists produces symptoms in healthy controls that closely mimic those found in schizophrenic patients (Krystal et al., 1994). Further, when stable schizophrenic patients are given ketamine, an NMDA antagonist and PCP analog, their symptoms are exacerbated and they experience similar clinical manifestations to their pre-medicated state (Lahti et at,, 1995b; Lahti et al, 1995a). We recently demonstrated the sensitivity of 11 C-raclopride binding to changes in human brain activity induced by ketamine (Smith et al., 1998), while simultaneously collecting clinical measures of psychosis. Our results indicated that acute administration of ketamine decreased 11C-raclopride binding, thus increasing synaptic dopamine activity while concurrently producing psychosis. Our investigation, and the strategy of using 11C-raclopride binding to measure glutamatergic induced changes in striatal dopamine, was later supported by Breier et al., who found decreased 11C-raclopride binding in healthy subjects after ketamine administration (Breier et al., 1998). Figure 2 indicates the time activity of basal receptor occupancy by 11C-raclopride and subsequent receptor occupancy after 1.0 mg/kg PCP in both the striatum and cerebellum. It is clear by the difference in striatal receptor occupancy (circles), that PCP administration diminishes HC-raclopride binding compared to baseline, indicative of increased competition by synaptic dopamine release secondary to PCP. Additionally, the difference in cerebellar Da occupancy between pre- and post-challenge is clearly minimal (triangles), justifying its use as a reference region in kinetic models. Figure 2. Time activity curve from a baseline 11C-racIopride scan (filled) and after PCP administration (open) 0.020 0.018 :

_gg»*

0.014 -

% Dose/cc

m

4*

0.016 -

_

^o wV ^ v x

0.012 0.010 -

3

0.008 : »

0.006 :

• O v v

o o o

Baseline Striatum Post Challenge Striatum Baseline Cerebellum Post Challenge Striatum

—

•

, 9

o

*V ^

o

^T

• 0

v

v

V

v

0.004 :

•

°

Tf

O

°

T

v ?

0.002 :

o

' '

A nnn

u.UUU

c

10

20

30

40

50

60

Time (min)

We have since taken this strategy one step further and demonstrated that decreased "C-raclopride binding subsequent to NMD A-antagonist administration can be successfully modulated with GABAergic pretreatment. In other words, increased dopaminergic activity by PCP administration was diminished by

614

HANDBOOK OF RADIOPHARMACEUTICALS

pretreatment with GVG, as evidenced by restoration of " C-raclopride binding to levels similar to the test/retest group (Schiffer el a/., submitted). Table 2 provides the change in mean distribution volume ratio (DVR) from the baseline scan (11C-raclopride alone) and the post-challenge scan ("C-raclopride in the presence of treatment paradigms) indicated. Lower numbers indicate less 11C-raclopride binding and higher endogenous dopamine receptor occupancy, while higher numbers indicate more 11C-raclopride binding as a result of less competition from synaptic dopamine. Further, changes in striatal dopamine for one primate given PCP Alone and one given GVG prior to PCP pretreatment are presented in Figure 3. Here, it is clear that there is less "C-raclopride binding in the primate given PCP, whereas in the animal pretreated with GVG, "C-raclopride binding in the second scan is very similar to baseline.

Table 2. Change in mean Distribution Volume Ratio (DVR) from baseline

scan to post-challenge scan % Change in Mean DVR

Group

7 ±1.2

T/RT

GVG Alone

18.8 ±3.2

PCP Alone

•3315.31

GVG + PCP

12 ±8.42

Figure 3. Logan plots of the striatal distribution volume from a non-human primate given PCP Alone (a) or an animal pretreated with GVG and given a PCP Challenge (b).

a. PCP Alone

b. GVG + PCP

Baseline

100

PCP 80

o (E

>

60

2 O

s° 20

20

60 80 40 /0TCpdt/ROI(T)

100

20 40 60 80 100 120 140 ATCpdt/ROI(T)

DYNAMIC NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET

615

This investigation demonstrated that it is possible to modulate glutamate-induced increases in dopamine with increased inhibitory GABA levels, and that this pharmacologic interaction can be measured with HCraclopride binding and PET. This study is supported by our dialysis investigations employing a similar paradigm by measuring changes in extracellular dopamine release after GVG pretreatment and a PCP challenge (Schiffer et al., submitted). Together, these investigations support the utility of GVG, in combination with neuroleptics, as a possible therapeutic treatment for schizophrenic psychoses. It has been demonstrated that GVG successfully ameliorates many of the extra-pyramidal side effects secondary to chronic neuroleptic treatment (Korsgaard et al., 1983; Thaker et al., 1983). Additionally, it has been demonstrated that patients receiving long-term neuroleptic therapy are more vulnerable to psychostimulant abuse (Roberts & Vickers, 1984; Roberts & Vickers, 1987; Brady et A3., 1990), so the anti-addictive properties of GVG demonstrated here might prove additionally therapeutic in schizophrenic populations. Finally, combining GVG with neuroleptic therapy might enable clinicians to diminish the dose of both drugs necessary for therapeutic efficacy (Wassef et al., 1999). MODULATION OF CHOLINERGIC ACTIVITY STUDIED WITH 11C-BENZTROPINE The function of the cholinergic system and its modulation by other systems may be critical to the pathophysiology of Alzheimer's disease and schizophrenia, and may provide insight into the manifestation of extrapyramidal side effects produced by antipsychotic treatment. An example of a relevant application of this approach is to study Alzheimer's disease. Alzheimer's disease has classically been characterized by a cortical cholinergic deficit (Bartus et al., 1982). The majority of drug trials have used therapeutic agents (e.g., cholinergic agonists and cholinesterase inhibitors) that were designed to directly reverse this deficiency. With few exceptions, these drugs have not been efficacious. Further, a model for schizophrenia has been proposed that hinges on cholinergic/dopaminergic interactions. In this model, cholinergic hyperactivity is postulated as an adaptive response to presumed dopaminergic hyperactivity and is considered to be responsible for the negative symptoms of schizophrenia (Tandon & Greden, 1989). More recent investigations support the involvement of muscarinic receptors specifically, in the pathogenesis of schizophrenia (Crook et al., 2000). It has been difficult to tease out the contributions of the disease mechanism itself, or of the effects of neuroleptic medications on schizophrenic pathology. Side effects associated with neuroleptic treatment (tardive dyskinesia) are alleviated by treatment with anticholinergic drugs (Lewis, 1998). These investigations provided the clinical impetus for investigating the cholinergic system over other neurotransmitter systems. Prior to addressing the usefulness of PET for investigating intrinsic interactions between ACh and DA, it was essential to develop a radiotracer specific for the cholinergic receptor. We chose benztropine for several reasons. First, it is a clinically prescribed synthetic anticholinergic drug frequently used in conjunction with neuroleptic medication in humans. Benztropine rapidly alleviates the extrapyramidal side effects commonly associated with dopaminergic D2 receptor blockade. Secondly, benztropine has a long biological half-life, which suggested it remained intact in the systemic circulation. Thirdly, it could be radiolabeled with carbon11 in high specific activity (0.75 - 2.0 Ci/umol). Recently, the ligands 11C-tropanyl benzilate and 11C-Nmethyl-4-piperidyl benzilate have been developed as radiotracers for the muscarinic receptor (Mulholland et

616

HANDBOOK OF RADIOPHARMACEUTICALS

al., 1992; Koeppe et al., 1992; Lee et al., 1991). The primary advantages of 11C-benztropine over other muscarinic cholinergic ligands is that pharmacologic doses can be administered to humans, so that the extent of specific binding in the PET data, using the unlabeled compound, can be determined. Additionally, striatal DV values obtained with these other radiotracers (11C-tropanyl benzilate and 11C-N-methyl-4-piperidyl benzilate) are consistent with our 11C-benztropine results. It is important to note that benztropine has been shown to inhibit DA reuptake in the corpus striatum in vitro (Coyle & Snyder, 1969). In order to determine which component of the PET image of labeled benztropine was due to binding to the DA transporter, we pretreated animals with nomifensine (2.0 mg/kg), a potent DA transport blocker (Dewey et al., 1990b). Incorporation of labeled benztropine was not altered in any brain structure examined following nomifensine pretreatment. Subsequent studies were performed with 11C-benztropine and GBR-12909 and 11Cbenztropine and cocaine in order to examine whether or not labeled benztropine was binding to the DA transporter in the striatum of the baboon brain. GBR-12909 was chosen as we have demonstrated mat it decreases the striatal binding of labeled raclopride presumably due to its binding to the DA transporter. Cocaine was chosen for similar reasons. Systemic administration of either GBR-12909 or cocaine did not alter the binding of labeled benztropine in any brain region examined. These studies are consistent with the nomifensine data. Finally, unlabeled benztropine did not alter 11C-cocaine binding. It appears that the binding of 11C-benztropine to the DA transporter does not make a significant contribution to the PET image (Dewey et al., 1990b). Cholinergic cells in the striatum only represent a small population of striatal neurons, but are able to modulate the excitability in this brain region due to their widespread axonal fields and high sensitivity to related neurotransmitter systems (Kincaid et al., 1998). Studies investigating in vivo and in vitro acetylcholine release have demonstrated that dopamine controls cholinergic transmission in a facilitory manner both directly and indirectly (Damsma et al., 1990). For a review of our studies with the cholinergic ligand, 11C-benztropine, please see Table 3.

Table 3. Effects of neurotransmitter-specific pharmacological challenges on cholinergic activity measured with PET Neurotransmitter System

Radioligand

Drug Challenge

Challenge Effect

Radioligand Response

Cholinergic Activity

Dopamine

11

C-benztropine

NMSP

£ DA

£

ft

Serotonin

11

C-benztropine

altan serin

0 5-HT

$

ft

GABA

11

C-benztropine

vigabatrin

ft GABA

&

ft

DYNAMIC NEUROTRANSMITTER INTERACTIONS MEASURED WITH PET

617

DOPAMINERGIC MODULATION OF ACETYLCHOLINE Our initial studies of the cholinergic system as the outcome measure for choline/dopamine interactions explored the effects of decreased dopaminergic activity on

11

C-benztropine binding. We first studied the

11

effect of a doparnine antagonist on the binding of the C-benztropine in the baboon (Dewey et al., 1990a). Pretreatment with unlabeled NMSP, a potent dopaminergic antagonist, reduced 11C-benztropine binding in all brain regions, with the effects in the corpus striatum being greater than the cortex and thalamus, and very little binding found in the cerebellum. Our data are consistent with a physiology where cholinergic interneurons are disinhibited from blockade of D2 afferents (Bymaster et al., 1986), producing increased acetylcholine release and subsequently decreased 11 C-benztropine binding. Here, we demonstrated decreasing dopaminergic activity with NMSP produced subsequent increases in acetylcholine levels, indexed by decreases in 11C-benztropine binding (Table 3). SEROTONERGIC MODULATION OF ACETYLCHOLINE The modulatory role of serotonin on acetylcholine activity has been extensively documented (Giovannini et al., 1998)), and more recent theories propose serotonin stimulates acetylcholine release through increased dopaminergic activity in animals, consistent with our primate investigations (Ramirez et al., 1997). The dorsal and median raphe nuclei are the major sources of 5-HT in the CNS. 5-HT2 receptors have been localized to cortical cholinergic nerve terminals as revealed by the loss of these receptors secondary to lesions of the nucleus basalis of Meynert (Quirion & Richard, 1985). Serotonergic enervation to the striatum is derived from the dorsal and medial raphe nuclei, which project to the striatum, pallidum, and substantia nigra (mainly the pars compacta). Serotonin has been shown to inhibit striatal ACh release (Euvrard et al., 1977; Guyenet et al., 1977) and depletion of endogenous 5-HT increased the release of striatal ACh (Visi et al., 1981). Gillet and coworkers (Gillet et al., 1985) demonstrated that exogenously administered 5-HT, 5HT agonists, or fluoxetine, an inhibitor of 5-HT uptake, reduced striatal ACh release, whereas methylsergide, a 5-HT agonist, increased ACh efflux in the caudal striatum (Jackson et al., 1988). However, Robinson (Robinson, 1983) found no effect of 5-HT on striatal ACh levels but instead reported an inhibition of ACh in cortex and hippocampus. We examined the modulation of acetylcholine by serotonin by measuring the effect of the serotonin antagonist altanserin on the binding of ''C-benztropine in primates (Dewey et al., 1993a). Decreasing serotonin activity with altanserin led to a decrease of striatal benztropine binding of ~30%; which is consistent with profound regional increases in acetylcholine release. These results confirm data from other studies indicating serotonin exerts a predominantly inhibitory influence on cholinergic interneurons in the rat striatum (Visi et al., 198]). In sum, decreasing serotonin activity with altanserin produced increases in synaptic acetylcholine levels, measured by decreases in C-benztropine binding.

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GABAergic MODULATION OF ACETYLCHOLINE Due to the large inhibitory capacity of the GABA system, many investigations have focused on the effects of GABAergic agents on acetylcholine release. The ventral pallidum receives direct input from ventral striatal regions that contain a large number of GABAergic interneurons (Heimer & Wilson, 1975). It is unresolved, however, whether this GABAergic input originates from these striatal GABAergic neurons or whether it arises from axons of other projection or GABAergic interneurons. Unlike the findings reported with 5-HT, studies with GABA and GABA mimetic drugs such as muscimol and SL 76 002 have demonstrated the ability to increase striatal ACh content, the largest of which was observed in the rat striatum with smaller effects in the cortex, nucleus accumbens, olfactory tubercle, hippocampus, interpendicular nucleus, hypothalamus and brainstem (Scatton & Bartholini, 1980). GABAergic neuronal terminals make contact not only with cholinergic neurons (DeBoer & Westerink, 1994), but also with glutamatergic (Moratalla & Bowery, 1991) and dopaminergic neurons in the striatum (Bowery et a/., 1990). Furthermore, striatal cholinergic neurons are regulated by glutamatergic and dopaminergic neurons, which are thought to form synapses on cholinergic neurons. It is thought that striatal GABAergic inhibition of dopaminergic or glutamatergic activity relieves the tonic inhibition or excitation of cholinergic neurons (Ikarashi et al., 1998; Moratalla & Bowery, 1991). A recent microdialysis study demonstrated that the GABAergic system appears to inhibit tonically the output of striatal acetylcholine via GABAA receptors, but not via GABAB receptors (DeBoer & Westerink, 1994). GVG provides an ideal mechanism for measuring the effects of increased endogenous GABA activity on the cholinergic system, as its effects are not mediated through any specific receptor system. Investigating the effects of GVG on the regional binding of "C-benztropine in the primate brain produced interesting results that supported the utility of PET as a measure of in vivo neurotransmitter activity (Dewey et al, 1993a). GVG produced a regionally specific decrease in 11C-benztropine binding. Striatal binding decreased 47% and cortical binding decreased 28%, but no changes in either thalamic or cerebellar uptake were observed. These regional and quantitative changes are consistent with the aforementioned excitatory role of GABA in striatal and cortical acetylcholine release (Scatton & Bartholini, 1980). Taken with our previous work using 11C-raclopride and GVG (Dewey et al., 1992b), this study represents the first demonstration with PET that a single drug (GVG), within the same time frame, can produce opposite effects in different neurotransmitter systems within the same animal. Specifically, GVG administration increased 11C-raclopride binding and decreased 11C-benztropine binding. Increasing GABA activity with vigabatrin produced subsequent increases in cholinergic activity, measured by decreases in 11C-benztropine binding. SUMMARY These data support the use of PET not only to monitor changes in synaptic neurotransmitter concentrations, but also to assessing the multiple mechanisms of action of new and potentially useful centrally acting therapeutic drugs. These findings have implications for the pathophysiology and pharmacotherapy of disease states that have classically been defined as neurotransmitter specific in origin. Specifically, we can use this

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application of PET as a tool to determine whether the ability of a neurotransmitter to modulate the activity of another functionally linked neurotransmitter is involved in the disease process. By capitalizing on our knowledge concerning neurotransmitter interactions, this would have direct implications for treatment in that therapeutic efficacy could be achieved indirectly, rather than directly, altering neurotransmitter activity. Potential treatments could be developed for cholinergic, dopaminergic and serotonergic defect states. Furthermore, this information may be used to predict the potential side effects of pharmacologic treatment in psychiatric disorders. Combined with an exhaustive literature supporting the fundamental principle that neurotransmitters interact in both functionally-specific and regionally-specific neuroanatomical foci, it is becoming increasingly clear that new treatment strategies for brain disorders (including addictions to cocaine, nicotine, heroin, and memamphetamine) should be developed with a more global awareness of this fundamental and welldocumented principle. While changes in individual neurotransmitter concentrations may indeed underlie the etiology of a specific disorder, it is likely that disease progression and symptom development are linked to compensatory or disease-induced changes in other neurotransmitters functionally linked to the original target. With this knowledge, we have been developing novel treatment strategies specifically designed to alter one or more neurotransmitters by targeting another. Our findings with nicotine, cocaine, methamphetamine, alcohol and GVG represent the potential utility of such a fundamental approach. ACKNOWLEDGMENTS This research was carried out at Brookhaven National Laboratory under contract with the U.S. Department of Energy, Office of Biological and Environmental Research (USDOE/OBER DE-AC02-98CH10886) and by the National Institutes of Mental Health (NIMH MH49165 and NIMH R2955155) and the National Institute on Drug Abuse (5RO-DA06278).

FOOTNOTES 1 Given a five-fold higher affinity than 11C-raclopride, our laboratory demonstrated in vivo sensitivity of 18FNMSP to dopamine in PET studies performed following d-amphetamine administration. These studies demonstrated that in vivo pharmacokinetic effects such as tissue clearance play an important role in radioligand sensitivity of high affinity radioligands to endogenous DA concentrations (Dewey et al., 1993; Logan et al., 1991). 2

In primates, the accumbens cells blend with those of the anteroventral putamen and the ventral caudate, so that a distinct border of the accumbens is not evident (Heimer et al., 1991). Recently, Drevets et al., have demonstrated that any potential bias effects due to resolution of PET cameras was significantly smaller than the magnitude of the observed changes in 11C-raclopride binding after amphetamine administration, supporting the correlation between microdialysis studies of psychostimulant activity in the NAcc and PET studies of psychostimulants in primates (Drevets et al., 1999).

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753. Thaker GK, Hare TA and Tamminga CA (1983) GAB A system: clinical research and treatment of tardive dyskinesia. Modern Problems of Pharmacopsychiatry, 21, 155–167. Tiihonen J, Kuoppamaki M, Nagren K, Bergman J, Eronen E, Syvalahti E and Hietala J (1996) Serotonergic modulation of striatal D2 dopamine receptor binding in humans measured with positron emission tomography. Psychopharmacology, 126, 277-80. Tsukada H, Nishiyama S, Kakiuchi T, Ohba H, Sato K and Harada N (1999) Is synaptic dopamine concentration the exclusive factor which alters the in vivo binding of [11C]raclopride?: PET studies combined with microdialysis in conscious monkeys. Brain Research, 841, 160–169. Tune LE, Wong DF, Pearlson G, Strauss M, Young T, Shaya EK, Dannals RF, Wilson AA, Ravert HT, Sapp J (1993) Dopamine D2 receptor density estimates in schizophrenia: a positron emission tomography study with "C-N-methylspiperone. Psychiatry Research, 49, 219-237. Visi ES, Harsing LGJ and Zsilla G (1981) Evidence of the modulatory role of serotonin in acetylcholine release from striatal interneurons. Brain Research, 212, 89–99. Volkow ND, Fowler JS and Wang GJ (1999) Imaging studies on the role of dopamine in cocaine reinforcement and addiction in humans. J. Psychopharm., 13, 337-345. Vollenweider FX, Vontobel P, Hell D and Leenders KL (1999) 5-HT modulation of dopamine release in basal ganglia in psilocybin-induced psychosis in man—a PET study with [11C]raclopride. Neuropsychopharmacology, 20, 424–433. Wassef AA, Dott SG, Harris A, Brown A, O'Boyle M, Meyer WJr and Rose RM (1999) Critical review of GABA-ergic drugs in the treatment of schizophrenia. J. Clin. Psychopharm., 19, 222-232.

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Weinberger DR (1997) The biological basis of schizophrenia: new directions. J. Clin. Psych., 58, Suppl 10, 22-27. Wesemann W, Weiner N, Rotsch M and Schulz E (1983) Serotonin binding in rat brain: circadian rhythm and effect of sleep deprivation. J. Neural Trans. Suppl., 18, 287–294. Wong DF, Wagner HNJ, Tune LE, Dannals RF, Pearlson GD, Links JM, Tamminga CA, Broussolle EP, Ravert HT, and Wilson AA (1986) Positron emission tomography reveals elevated D2 dopamine receptors in drag-naive schizophrenics [published erratum appears in Science 1987 Feb 6;235(4789):623. Science, 234, 1558-1563. Xi ZX and Stein EA (2000) Increased mesolimbic GABA concentration blocks heroin self-administration in the rat. J. Pharm. Exper. Ther., 294, 613–619. Zazpe A, Artaiz I and Del Rio J (1994) Role of 5-HT3 receptors in basal and K(+)-evoked dopamine release from rat olfactory tubercle and striatal slices. Brit. J. Pharm., 113, 968-972.

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22. TUMOR IMAGING ROLAND HUSTINX1 AND ABASS ALAVI2 1

Division of Nuclear Medicine, University Hospital of Liege, Sart Tilman B35, 4000 Liege, BELGIUM, 2Division of Nuclear Medicine, Hospital of the University of Pennsylvania, Donner Bldg Room 109, 3400 Spruce St, Philadelphia, PA 19104, USA

INTRODUCTION In recent years, FDG-PET imaging has become a major tool for diagnosing, staging, restaging and monitoring various malignant conditions. FDG-PET is an imaging technique that allows a unique approach for the assessment of the degree and the extent of cancer which is complementary to other techniques. Alterations in glucose metabolism, which are commonly noted in most cancers as revealed as functional images, FDG-PET provides information that may be distinct from that obtained by conventional imaging methods. As a result of considerable technological advances made in data acquisition and processing, high quality whole-body images can be obtained in a time frame which is comparable to other diagnostic techniques. Although spatial resolution of PET is inferior to that of CT or MR, it reflects metabolic phenomena that take place at the molecular level. PET provides images which reveal cellular or intracellular biochemical processes that are dependent on the presence of specific enzymes or membrane receptors. FDG is by far the most widely used radiotracer for clinical purposes, but other radiopharmaceuticals are being actively investigated, including tracers for amino acid uptake, cell proliferation and tumor hypoxia. The ability to evaluate metabolic alterations in tumor cells prior to any detectable anatomical or structural changes results in a significant impact in patient management and substantially improves our understanding of the evaluation of the disease processes over time. FOG and glucose metabolism FDG is a glucose analog and as such it enters the cells using the same membrane transporters as glucose. Both FDG and glucose are phosphorylated by hexokinase but unlike glucose-6-P, FDG-6-P is not a substrate for further metabolism in the glycolytic pathway. Since the levels of glucose-6-phosphatase are very low in benign and malignant tissues, dephosphorylation of FDG-6-P into FDG is very slow and limited. FDG-6-P therefore accumulates inside the cells, in proportion to their glycolytic activity. Various normal tissues take up FDG, in particular, gray matter in the brain. Variable uptake may be seen in skeletal muscle, the digestive tract, especially the stomach and colon, salivary glands, laryngeal muscles, the thyroid and (in young patients) the thymus. Although patients routinely fast for at least 6 hours before the study in order to shift the myocardial metabolism to fatty acids, significant heart uptake is seen in up to 30% of patients. A significant fraction of the administered FDG is mostly excreted through the kidneys (Cook et al, 1996; Shreve et al, 1999). Figure 1 shows a normal FDG distribution.

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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Figure 1. Normal FDG PET study. Note the renal excreation of the tracer, the cardiac uptake and the moderate moderate liver and spleen uptake. As published by Warburg, glucose metabolism is enhanced in most tumor cells (Warburg et al, 1930; Warburg, 1956). Three phenomena are primarily responsible for increased glucose utilization in tumor cells: overexpression of genes coding for glucose membrane transporters, in particular GLUT 1 and to a lesser extent GLUT 3 (Haberkorn et al., 1994; Higashi et al., 1997), increased hexokinase activity and decreased levels of glucose-6-phosphatase (Pauwels et al., 1998). The increase in glucose phosphorylation is related to the presence of type II hexokinase at the mitochondrial surface. This subtype of the enzyme is extremely active and does not participate in normal glycolysis. It must be noted that the relative contribution of these phenomena varies from one tumor to the other. For instance, it has been showed that increased FDG uptake in breast tumors is primarily due to increased levels of GLUT1, and the rate limiting factor is hexokinase-driven phosphorylation, whereas is non-small cell lung cancers, hexokinase is very active and does not constitute a limiting factor (Torizuka et al., 1998). Although most tumor types display increased glucose uptake, the underlying reasons for this is still subject to debate. In most instances, it is generally accepted that FDG uptake is primarily related to the number of viable tumor cells and/or their increase in transcription of genes coding for the regulation of the glucose metabolism (Higashi et al., 1993; Haberkorn et al., 1994; Higashi et al., 1998; Brown et al., 1999). For some tumor cell types, however, data suggest a direct relationship between FDG uptake and rate of cell proliferation (Duhaylongsod et al., 1995; Okada et al., 1992; Higashi et al., 2000). In addition, FDG uptake is not homogeneously distributed throughout the tumor. Within a lesion, there is a good correlation between FDG uptake and regional distribution of GLUT1, as shown by autoradiographic studies in animals (Brown et al., 1996). While necrotic parts of the tumor do not take up FDG, cell hypoxia significantly enhances it, partly due to a further increase in GLUT1 expression (Clavo et al., 1995; Clavo & Wahl, 1996; Minn et al., 1996). FDG also accumulates in inflammatory cells, activated macrophages and polymorphonuclear leukocytes, and it has been demonstrated that granulomas and granulation tissues reveal high levels of glucose metabolism (Yamada et al., 1995). In most tumors, however, increased uptake is primarily in the tumor cells, and inflammatory and granulation cells are responsible for a fraction of noted metabolic activity (Brown et al., 1995).

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In addition, kinetics of FDG vary greatly among different tissue types. In malignant tumors, the maximum uptake is reached relatively late after FDG injection, up to 6 hours, while in inflammatory masses and benign tumors a plateau level is noted within 45 to 60 minutes (Fischman & Alpert, 1993; Lodge et al., 1996). This can be of some value in differentiating malignant and benign lesions and thus increase the specificity of the test (Hustinx et al., 1999). Based on experience gained over the past decade, FDG-PET imaging usually starts 60 minutes after injection, so that a satisfactory tumor to background activity ratio is achieved. Non-specific uptake of FDG in inflammatory sites has led investigators to explore the role of this methodology for the detection of infection and related processes (Guhlmann et al., 1998; Sugawara et al., 1998). When FDG-PET imaging is utilized to examine patients with cancer at various stages of the disease, it is extremely important that the numerous pitfalls that can lead to false positive results are taken into consideration (Strauss, 1996). In particular, prior diagnostic (biopsies) or therapeutic interventions (surgery) are known to result in significant uptake of FDG. A frequently encountered and a potentially confounding factor is radiation therapy which increases glucose metabolic uptake early after treatment and may persist for prolonged periods of time (Furuta et al,, 1997; Hautzel & Muller-Gartner, 1997; Higashi et al., 1993).

ONCOLOGICAL APPLICATIONS OF FDG-PET IMAGING DIAGNOSIS FDG-PET imaging is performed as a diagnostic test in patients with solitary pulmonary nodules (SPN), undefined pancreatic masses, in some selected cases of breast cancers and possibly in other malignancies where conventional methods have failed to establish the diagnosis. PET is highly effective in characterizing SPN, with a sensitivity of over 90% and a relatively high specificity, which may vary depending on the prevalence of the known causes of false positive results (i.e., tuberculosis, histoplasmosis, blastomycosis), etc., (Lowe et al, 1998; Patz et al., 1993; Bury et al., 1996). FDG-PET imaging appears to be superior to transthoracic needle biopsy (CT guided) in establishing the accurate diagnosis in SPN. Dewan et al. reported superior performance for PET without the significant morbidity associated with such biopsies (Dewan et al., 1995). A recent cost-effectiveness analysis reported from Europe demonstrated that a PET-based strategy may lead to an increase in life expectancy at a very low cost (Dietlein et al., 2000). PET is also used for differentiating mass-forming pancreatitis from cancer (Zhnny et al, 1997; Imdahl, et al., 1999). In these patients, knowledge of the blood glucose level is of particular importance as the sensitivity decreases with hyperglycemia at the time of the examination (Diederichs et al., 1998). False positive results have also been reported in cases of acute pancreatitis (Shreve, 1998). FDG-PET imaging has been proposed as an adjunct method for diagnosing breast cancers in selected cases, such as in patients with radiographically dense breasts, in patients with implants and in cases of failed biopsy for definitive tissue characterization (Wahl et al., 1994; Scheidhauer et al., 1996). Examples of FDG-PET in diagnosis are given in Figures 2 and 3.

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Figure 2: Newly diagnosed non-small cell lung cancer. FDG-PET showed hypermetabolic pratracheal and hilar lymph nodes (figure 2A and 2B; coronal slice, 2C and 2D; transverse slices). CT demonstrated the lung tumor, but failed to detect any nodal involvement (figure 2E and 2F). Surgery confirmed PET findings: adenocarcinoma, stage III.

Figure 3: Peritoneal recurrence of a colon carcinoma. FDG-PET was performed because of an elevation of CEA levels, the conventional work up, including abdominopelvic CT, was negative.

STAGING Patients with cancer cannot be appropriately managed without accurate staging. This is perhaps best exemplified by non-small cell lung cancer (NSCLC). Surgery is the only possible curative treatment and survival of patients with NSCLC depends on the stage of their disease which include the state of the mediastinal lymph nodes at the time of diagnosis. Unlike CT, which solely relies upon the size of the lymph nodes to diagnose metastatic involvement, FDG-PET imaging reveals direct evidence for such diagnoses and there is now a large body of evidence demonstrating the superiority of PET in NSCLC over any other imaging method (Bury et al., 1997; Gupta et al., 1999; Vansteenkiste et al., 1998). In these patients, improved sensitivity and specificity fully translate into making decisions about the management plans, including the selection of those who will benefit from a major surgery. FDG-PET

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imaging is therefore a highly cost effective method for preoperative staging of NSCLC and has become part of the routine work-up of patients with NSCLC in a growing number of centers (Gambhir et al, 1996; Scott et al., 1998; Kosuda et al, 2000; Dietlein et al, 2000). PET can also be useful in the preoperative staging of various cancers such as esophagus (Flamen et al, 2000), pancreas (Frohlich et al, 1999), uterus (Rose et al, 1999), and sarcomas (Lucas et al, 1999). FDG-PET imaging has been proposed as a method for detecting nodal involvement in primary breast cancer. It performs relatively well for this purpose, with the ability of visualizing tumor spread to both axillary and internal mammary nodal stations, as well as distant metastases (Avril et al, 1996; Adler et al, 1997). An example of nodal occurrence detected by the FDG is shown in Figure 4.

Figure 4: Nodal recurrences of a breast carcinoma, previously treated by surgery, irradiation and chemotherapy. These very small lesions are clearly visualized on the FDG-PET study. However, PET is not widely accepted as a possible replacement for surgical axillary lymph node dissection. The acceptable threshold for the negative predictive value remains unclear, since most patients with small metastases may go unnoticed with this technique. Similarly, despite its high sensitivity and specificity (Adams et al, 1998), FDG-PET imaging may not be cost-effective for staging primary head and neck cancers (Hanasono et al, 1999). For melanomas, and possibly for breast cancers, FDG-PET imaging should be employed in conjunction with sentinel lymph node biopsy, as it lacks sensitivity to detect microscopic involvement (Wagner et al, 1999). On the other hand, it is very effective for screening distant metastases in high-risk patients (i.e. with high Breslow index) (Boni et al, 1995; Paquet et al, 2000). Finally, strong data indicate that FDG-PET is a cost-effective method for staging high-grade non-Hodgkin lymphomas at the time of initial diagnosis and during the course of the disease (Klose et al, 2000). It constitutes an excellent base for the follow up of such patients who will receive several courses of chemo- and/or radiotherapy (Jerusalem et al, 1999). FDG-PET imaging is much more effective than 67Ga and therefore, is the modality of choice for managing patients with lymphoma (Kostakoglu & Goldsmith, 2000). DIAGNOSIS AND STAGING OF RECURRENT DISEASE Identifying, locating and defining the exact extent of a recurrent tumor remains a challenging task. In many cases, conventional imaging methods such as CT and MR, cannot distinguish fibrous scar and other inactive tissues from recurrent tumors. Irradiation and surgery often greatly alter the anatomy,

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rendering structural imaging techniques unable to detect persistent or recurrent disease. Because of its ability to determine disease activity, FDG-PET imaging can be proposed as the primary imaging modality for several situations. Suspected recurrent colorectal cancer, because of an elevated CEA level, with a negative conventional work-up is a major indication for FDG-PET imaging. In this case, PET can most often either localize the relapse or exclude it, leading to significant changes in the management of these patients (Flanagan et al., 1998). In cases of suspected recurrence of colorectal cancer with equivocal findings with conventional methods and in patients with proven relapse who are selected for curative surgery, FDG-PET imaging has proven to be extremely effective, in optimizing therapy, and therefore being employed as the modality of choice in such settings. (Valk et al., 1999; Fong et al., 1999; Delbeke et al., 1997; Flamen et al., 1999). The situation is similar in patients with a previous history of head and neck carcinoma (Farber et al., 1999), and ovarian carcinoma (Hubner et al., 1999) where conventional imaging methods perform generally very poorly. Lymphoma patients often present with persistent masses on CT after completion of chemotherapy. Whether these masses represent viable tumor or fibrosis is of primary importance, since such determination is critical for forecasting the overall survival. PET is now established as the study of choice to decide whether a patient has responded to treatment, and therefore assists the clinician for course of action (Jerusalem et al., 1999; Bangerter et al., 1999). For almost a decade, PET has been used to distinguish recurrence of brain tumors from radiation necrosis. Non-specific contrast enhancement is frequently observed on MRI, in both conditions and therefore, a definitive diagnosis is often made based on determination of metabolic activity of the specified lesion (Hustinx & Alavi, 1999). EVALUATION OF THE RESPONSE TO TREATMENT This may represent the most important application of PET imaging in the future and may substantially influence the management of patients with cancer. Current methods are purely based on assessment of structural changes following treatment, in particular size of the lesions, as determined by anatomical methods (CT, MRI, US, etc.) or physical examination. Metabolic response to chemo or radiation therapies, including changes in glucose metabolism, is noted soon after the treatment is initiated, and may be measured and quantified by PET imaging in advance of structural alterations seen on anatomic imaging techniques. Preliminary studies have yielded encouraging results, particularly patients with locally advanced breast cancers. Two independent groups recently showed that FDG-PET imaging performed as early as after a single course of chemotherapy, is a good predictor of the final therapeutic response (Smith et al., 2000; Schelling et al., 2000). One can easily imagine the potential impact of such findings on the management of these patients. Similarly encouraging results were obtained in other cancers but, as summarized by Eary et. al. in a recent editorial, tracers with higher specificity than FDG are probably better suited to reliably evaluate the response to treatment (Eary & Krohn, 2000). Protein synthesis and aminoacid uptake "C-methionine (MET) is used in some centers for brain tumor imaging. Unlike FDG, it is not taken up by the normal cortex, and therefore brain tumors are clearly delineated with MET than with FDG. MET uptake initially was thought to reflect the rate of protein synthesis, but it is also related to amino acid transport and altered amino acid metabolism (Ishiwata et al., 1993). Its uptake may also be influenced by blood-brain barrier alterations (Hatazawa et al., 1989; Roelcke et al., 1995). MET-PET is particularly useful to evaluate low-grade primary brain tumors and their response to treatment (Wurker et al., 19%; Kaschten et al., 1998). In patients with non-CNS cancers, whole-body imaging with MET-PET has not been shown to be superior to FDG-PET (Inoue et al., 1996). Other amino acid analogs are thus being

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developed, with encouraging preliminary results. L-[3-18F]-a-methyltyrosine (FMT), accumulates in tumor cells solely via an amino acid transport system (Inoue et al., 1998). It was recently shown to accumulate in high-grade musculoskeletal sarcomas and to be more effective than FDG in differentiating benign from malignant tumors (Watanabe et al., 2000). The new analog O-(2-[18 F]fluoroethyl)-L-tyrosine (FET) is transported by the L-amino acid transport system. It is not incorporated into proteins nor metabolized. The exact mechanism of uptake and its retention in tumors is not fully understood, but FET and MET studies performed in the same patient population with various brain tumor patients have yielded very similar results (Heiss et al., 1999; Wester et al., 1999; Weber et al., 2000). This indicates that FET, whose easy and high yield synthesis is established, may also have a potential for whole-body imaging, with the potential advantage of providing specific results than those noted with FDG-PET imaging in patients with suspected malignancy. Tumor proliferation Assessment of tumor proliferation is feasible using 11C-thymidine (Eary et al., 1999). However, accurate measurement of the rate of DNA synthesis requires extensive data processing and scanning protocols that can only be accomplished in a limited number of research centers (Mankoff et al., 1998; Mankoff et al., 1999). PET studies with 11C-thymidine demonstrated a reduction of the tumor proliferation in response to treatment, before any decrease in FDG metabolism, projecting a great potential for this type of tracer (Shields et al., 1998). This has led to the successful development of FLT ([F-18]3'-deoxy-3'fluorothymidine) (Shields et al., 1998). FLT is phosphorylated by human thymidine kinase but not further metabolized. Preliminary data indicate that its accumulation follows a 3-compartment model very similar to that of FDG, Although a good correlation between TK activity and FLT accumulation exists, it is not strong enough to fully account for the increased FLT accumulation and there must be other contributing phenomena to explain such uptake. Obviously, further investigation is needed to understand the uptake mechanism. Regardless of the mechanism, PET imaging with FLT provides high-quality images (Figure 5), the tracer is now readily available, and comparative studies with FDG are warranted to define its place in tumor imaging.

Figure 5: 18F-FLT PET study in a patient with disseminated lung cancer. In addition to the uptake in proliferating normal tissues such as bone marrow, several malignant lesions are seen in the right lung, liver and left sacroiliac joint. (Courtesy of B.M. Dohmen, M.D., University of Tuebingen)

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Hypoxia Tumor hypoxia is known to increase radioresistance and, for some tumors, chemoresistance. Higher rates of recurrence after surgical treatment has also been attributed to hypoxia in certain tumors. The level of hypoxia varies with tumor type, among patients, and within different lesions in a single patient PET imaging with 18F-fluoromisonidasole (18F-FMISO) has opened the way for the non-invasive assessment of tumor hypoxia. It has been used to quantify tumor hypoxia prior to treatment and to measure its evolution during the treatment (Rasey et al., 1989; Koh et al., 1995; Rasey et al., 19%). Other groups have developed additional compounds with potentially optimal imaging characteristics, attractive biodistributions and/or easier syntheses. These include imidazole derivatives, such as I8Ffluoroerythronitroimidazole (Yang et al., 1995), 18F-fluoroetanidazole (Tewson, 1997) and more recently 18 F-EF1 ([F-18]-N-(3-monofluoropropyl)-2-(2-nitroimidazol-l[H]-yl)-acetamide (Kachur et al., 1999). Encouraging results have been obtained in rodents with I8F-EF1 with a high tumor to background uptake ratio in hypoxic tumors, soon after tracer administration. No uptake was seen in normoxic tumors (Evans et al., 2000). Bis(thiosemicarbazone) derivatives labeled with copper isotopes, in particular 64Cu-ATSM (Dearling et al., 1998; Anderson et al., 1998; Lewis et al., 1999), are also being actively investigated. Retention of these agents in tumor cells depends primarily on a cytosolic/microsomal bioreduction, in particular via the NADH-cytochrome P450 reductase and NADH-cytochrome b5 reductase. Although the clinical utility of these agents remains unproven, the mere knowledge of such phenomena should impact positively on patient management in the foreseeable future. REFERENCES Adams S, Baum RP, Stuckensen T, Bitter K and Hor G (1998) Prospective comparison of I8F-FDG-PET with conventional imaging modalities (CT, MRI, US) in lymph node staging of head and neck cancer. Euro. J. Nucl. Med., 25(9): 1255–1260. Adler LP, Faulhaber PF, Schnur KC, Al-Kasi NL and Shenk RR (1997) Axillary lymph node metastases: screening with [F-18]2-deoxy-2-fluoro-D-glucose (FDG) PET. Radiology, 203(2): 323–327. Anderson CJ, Jones LA, Bass LA, Sherman EL, McCarthy DW, Cutler PD, Lanahan MV, Cristel ME, Lewis JS and Schwarz SW (1998) Radiotherapy, toxicity and dosimetry of copper-64-TETAoctreotide in tumor-bearing rats. J. Nucl. Med., 39( 11): 1944–1951. Avril N, Dose J, Janicke F, Ziegler S, Romer W, Weber W, Herz M, Nathrath W, Graeff H and Schwaiger M (19%) Assessment of axillary lymph node involvement in breast cancer patients with positron emission tomography using radiolabeled 2-(fluorine-18)-fluoro-2-deoxy-Dglucose. J. Nat'l. Cancer Inst., 88(17): 1204–1209. Bangerter M, Griesshammer M and Bergmann L (1999) Progress in medical imaging of lymphoma and Hodgkin's disease [see comments]. Cur. Op. Onc., 11 (5): 339–342. Boni R, Boni RA, Steinert H, Burg G, Buck A, Marincek B, Berthold T, Dummer R, Voellmy D and Ballmer B (1995) Staging of metastatic melanoma by whole-body positron emission tomography using 2-fluorine-18-fluoro-2-deoxy-D-glucose. Brit. J. Derm., 132(4):556-562. Brown RS, Leung JY, Kison PV, Zasadny KR, Hint A and Wahl RL (1999) Glucose transporters and FDG uptake in untreated primary human non-small cell lung cancer. J. Nucl. Med., 40(4):556-565. Brown RS, Leung JY, Fisher SJ, Frey KA, Ethier SP and Wahl RL (1996) Intratumoral distribution of tritiated-FDG in breast carcinoma: correlation between Glut-1 expression and FDG uptake. J. Nucl. Med., 37(6): 1042-1047. Brown RS, Leung JY, Fisher SJ, Frey KA, Ethier SP and Wahl RL (1995) Intratumoral distribution of tritiated fluorodeoxyglucose in breast carcinoma: I. Are inflammatory cells important? J. Nucl. Med., 36(10): 1854-1861.

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Bury T, Dowlati A, Paulus P, Corhay JL, Hustinx R, Ghaye B, Radermecker M and Rigo P (1997) Whole-body 18FDG positron emission tomography in the staging of non-small cell lung cancer. Euro. Resp. J., 10(11):2529-2534. Bury T, Dowlati A, Paulus P, Corhay JL, Benoit T, Kayembe JM, Limet R, Rigo P and Radermecker M (1996) Evaluation of the solitary pulmonary nodule by positron emission tomography imaging. Euro. Resp. J., 9(3):410-414. Clavo AC and Wahl RL (1996) Effects of hypoxia on the uptake of tritiated thymidine, L-leucine, Lmethionine and FDG in cultured cancer cells. /. Nucl. Med., 37(3):502-506. Clavo AC, Brown RS and Wahl RL (1995) Fluorodeoxyglucose uptake in human cancer cell lines is increased by hypoxia. J. Nucl. Med., 36(9): 1625–1632. Cook GJ, Fogelman I and Maisey MN (1996) Normal physiological and benign pathological variants of 18-fluoro-2-deoxyglucose positron-emission tomography scanning: potential for error in interpretation. Sem. Nucl. Med., 26(4): 308–314. Dearling JL, Lewis JS, Mullen GE, Rae MT, Zweit J and Blower PJ (1998) Design of hypoxia-targeting radiopharmaceuticals: selective uptake of copper-64 complexes in hypoxic cells in vitro. Euro. J. Nucl. Med., 25(7):788-92. Delbeke D, Vitola JV, Sandier MP, Arildsen RC, Powers TA, Wright JK, Jr., Chapman WC and Pinson CW (1997) Staging recurrent metastatic colorectal carcinoma with PET. J. Nucl. Med., 38(8): 1196–1201. Dewan NA, Reeb SD, Gupta NC, Gobar LS and Scott WJ (1995) PET-FDG imaging and transthoracic needle lung aspiration biopsy in evaluation of pulmonary lesions. A comparative risk-benefit analysis. Chest, 108(2): 441–446. Diederichs CG, Staib L, Glatting G, Beger HG and Reske SN (1998) FDG-PET: elevated plasma glucose reduces both uptake and detection rate of pancreatic malignancies. J. Nucl. Med., 39(6): 1030–1033 Dietlein M, Weber K, Gandjour A, Moka D, Theissen P, Lauterbach KW and Schicha H (2000) Costeffectiveness of FDG-PET for the management of solitary pulmonary nodules: a decision analysis based on cost reimbursement in Germany. Eur. J. Nucl. Med., 27(10): 1441–1456. Dietlein M, Weber K, Gandjour A, Moka D, Theissen P, Lauterbach KW and Schicha H (2000) Costeffectiveness of FDG-PET for the management of potentially operable non-small cell lung cancer: priority for a PET-based strategy after nodal-negative CT results. Eur. J. Nucl. Med., 27(11):1598–1609. Duhaylongsod FG, Lowe VJ, Patz EF, Jr., Vaughn AL, Coleman RE and Wolfe WG (1995) Lung tumor growth correlates with glucose metabolism measured by fluoride-18 fluorodeoxyglucose positron emission tomography. Annals of Thoracic Surgery, 60(5): 1348–1352. Eary JF and Krohn KA (2000) Positron emission tomography: imaging tumor response. Eur. J. Nucl. Med., (Published online: 23 August 2000). Eary JF, Mankoff DA, Spence AM, Berger MS, Olshen A, Link JM, O'Sullivan F and Krohn KA (1999) 2-[C-11]thymidine imaging of malignant brain tumors. Cancer Research, 59(3): 615–621. Evans SM, Kachur AV, Shiue CY, Hustinx R, Jenkins WT, Shive GG, Karp JS, Alavi A, Lord EM, Dolbier WR, Jr. and Koch CJ (2000) Noninvasive detection of tumor hypoxia using the 2nitroimidazole [I8F]EF1. J. Nucl. Med., 41(2):327-336. Farber LA, Benard F, Machtay M, Smith RJ, Weber RS, Weinstein GS, Chalian AA, Alavi A and Rosen thai DI (1999) Detection of recurrent head and neck squamous cell carcinomas after radiation therapy with 2-18F-fluoro-2-deoxy-D-glucose positron emission tomography. Laryngoscope, 109(6): 970-975.

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Fischman AJ and Alpert NM (1993) FDG-PET in oncology: there's more to it than looking at pictures [editorial; comment]. J. Nucl. Med., 34(1):6-11. Flamen P, Lerut A, Van Cutsem E, De Wever W, Peeters M, Stroobants S, Dupont P, Bormans G, Hiele M, De Leyn P, Van Raemdonck D, Coosemans W, Ectors N, Haustermans K and Mortelmans L (2000) Utility of positron emission tomography for the staging of patients with potentially operable esophageal carcinoma [see comments]. /. Clin. One., 18(18):3202-3210. Flamen P, Stroobants S, Van Cutsem E, Dupont P, Bormans G, De Vadder N, Penninckx F, Van Hoe L and Mortelmans L (1999) Additional value of whole-body positron emission tomography with fluorine-18-2-fluoro-2-deoxy-D-glucose in recurrent colorectal cancer. J. Clin. One., 17(3):894901. Flanagan FL, Dehdashti F, Ogunbiyi OA, Kodner LF and Siegel BA (1998) Utility of FDG-PET for investigating unexplained plasma CEA elevation in patients with colorectal cancer [see comments]. Annals of Surgery, 227(3):319–323. Fong Y, Saldinger PF, Akhurst T, Macapinlac H, Yeung H, Finn RD, Cohen A, Kemeny N, Blumgart LH and Larson SM (1999) Utility of 18F-FDG positron emission tomography scanning on selection of patients for resection of hepatic colorectal metastases. Am. J. Surg., 178(4):282287. Frohlich A, Diederichs CG, Staib L, Vogel J, Beger HG and Reske SN (1999) Detection of liver metastases from pancreatic cancer using FDG-PET. J. Nucl. Med., 40(2):250-255. Furuta M, Hasegawa M, Hayakawa K, Yamakawa M, Ishikawa H, Nonaka T, Mitsuhashi N and Niibe H (1997) Rapid rise in FDG uptake in an irradiated human tumour xenograft. Euro. J. Nucl. Med., 24(4):435–8. Gambhir SS, Hoh CK, Phelps ME, Madar I and Maddahi J (1996) Decision tree sensitivity analysis for cost-effectiveness of FDG-PET in the staging and management of non-small cell lung carcinoma [see comments]. J. Nucl. Med., 37(9): 1428-1436. Guhlmann A, Brecht-Krauss D, Suger G, Glatting G, Kotzerke J, Kinzl L and Reske SN (1998) Chronic osteomyelitis: detection with FDG-PET and correlation with histopathologic findings. Radiology 206(3):749-754. Gupta NC, Graeber GM, Rogers JS, 2nd and Bishop HA (1999) Comparative efficacy of positron emission tomography with FDG and computed tomographic scanning in preoperative staging of non-small cell lung cancer. Annals of Surgery 229(2): 286–291. Haberkorn U, Morr I, Oberdorfer F, Bellemann ME, Blatter J, Altmann A, Kahn B and van Kaick G (1994) Fluorodeoxyglucose uptake in vitro: aspects of method and effects of treatment with gemcitabine. J. Nucl. Med., 35(11): 1842–1850. Haberkorn U, Ziegler SI, Oberdorfer F, Trojan H, Haag D, Peschke P, Berger MR, Altman A and van Kaick G (1994) FDG uptake, tumor proliferation and expression of glycolysis associated genes in animal tumor models. Nucl. Med. Biol, 21 (6): 827–834. Hanasono MM, Kunda LD, Segall GM, Ku GH and Terris DJ (1999) Uses and limitations of FDG positron emission tomography in patients with head and neck cancer. Laryngoscope, 109(6):880-885. Hatazawa J, Ishiwata K, Itoh M, Kameyama M, Kubota K, Ido T, Matsuzawa T, Yoshimoto T, Watanuki S and Seo S (1989) Quantitative evaluation of L-[methyl-C-ll] methionine uptake in tumor using positron emission tomography. J. Nucl. Med., 30( 11): 1809–1813. Hautzel H and Muller-Gartner HW (1997) Early changes in fluorine-18-FDG uptake during radiotherapy. J. Nucl. Med., 38(9): 1384-1386.

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Heiss P, Mayer S, Herz M, Wester HJ, Schwaiger M and Senekowitsch-Schmidtke R (1999) Investigation of transport mechanism and uptake kinetics of O-(2-[18F]fluoroethyl)-L-tyrosine in vitro and in vivo. J. Nud. Med., 40(8): 1367-1373. Higashi K, Ueda Y, Yagishita M, Arisaka Y, Sakurai A, Oguchi M, Seki H, Nambu Y, Tonami H and Yamamoto I (2000) FDG-PET measurement of the proliferative potential of non-small cell lung cancer. J. Nucl. Med., 41(l):85-92. Higashi K, Clavo AC and Wahl RL (1993) Does FDG uptake measure proliferative activity of human cancer cells? In vitro comparison with DNA flow cytometry and tritiated thymidine uptake [see comments]. J. Nucl. Med., 34(3):414-419. Higashi K, Clavo AC and Wahl RL (1993) In vitro assessment of 2-fluoro-2~deoxy-D-glucose, Lmethionine and thymidine as agents to monitor the early response of a human adenocarcinoma cell line to radiotherapy [see comments]. J. Nucl. Med., 34(5):773-779. Higashi T, Tamaki N, Torizuka T, Nakamoto Y, Sakahara H, Kimura T, Honda T, Inokuma T, Katsushima S, Ohshio G, Imamura M and Konishi J (1998) FDG uptake, GLUT-1 glucose transporter and cellularity in human pancreatic tumors. J. Nucl. Med., 39(10): 1727–1735. Higashi T, Tamaki N, Honda T, Torizuka T, Kimura T, Inokuma T, Ohshio G, Hosotani R, Imamura M and Konishi J (1997) Expression of glucose transporters in human pancreatic tumors compared with increased FDG accumulation in PET study. J. Nucl. Med., 38(9): 1337–1344. Hubner KF, McDonald TW, Niethammer JG, Smith GT, Gould HR and Buonocore E (1993) Assessment of primary and metastatic ovarian cancer by positron emission tomography (PET) using 2[18F]deoxyglucose (2-[18F]FDG). Gyn. Onc., 51(2): 197-204. Hustinx R, Smith RJ, Benard F, Rosenthal DI, Machtay M, Farber LA and Alavi A(1999) Dual time point fluorine-18 fluorodeoxyglucose position emission tomography: a potential method to differentiate malignancy from inflammation and normal tissue in the head and neck. Eur. J. Nud. Med., 26(10): 1345–1348. Hustinx R and Alavi A (1999) SPECT and PET imaging of brain tumors. Neuroimaging Clin. N. Am. 9(4) :751–766. Imdahl A, Nitzsche E, Krautmann F, Hogerle S, Boos S, Einert A, Sontheimer J and Farthmann EH (1999) Evaluation of positron emission tomography with 2-[18F]fluoro-2-deoxy-D-glucose for the differentiation of chronic pancreatitis and pancreatic cancer. Brit. J. Surg., 86(2): 194–199. Inoue T, Tomiyoshi K, Higuichi T, Ahmed K, Sarwar M, Aoyagi K, Amano S, Alyafei S, Zhang H and Endo K (1998) Biodistribution studies on L-3-[fluorine-18]fluoro-alpha-methyl tyrosine: a potential tumor-detecting agent. J. Nud. Med., 39(4):663-667. Inoue T, Kim EE, Wong FC, Yang DJ, Bassa P, Wong WH, Korkmaz M, Tansey W, Hicks K and Podoloff DA (1996) Comparison of fluorine-18-fluorodeoxyglucose and carbon-11-methionine PET in detection of malignant tumors. J. Nucl. Med., 37(9): 1472–1476. Ishiwata K, Kubota K, Murakami M, Kubota R, Sasaki T, Ishii S and Senda M (1993) Re-evaluation of amino acid PET studies: can the protein synthesis rates in brain and tumor tissues be measured in vivo? J. Nucl. Med., 34(11): 1936–1943. Jerusalem G, Warland V, Najjar F, Paulus P, Fassotte MF, Fillet G and Rigo P (1999) Whole-body !8FFDG-PET for the evaluation of patients with Hodgkin's disease and non-Hodgkin's lymphoma. Nucl. Med. Comm., 20(1): 13-20. Jerusalem G, Beguin Y, Fassotte MF, Najjar F, Paulus P, Rigo P and Fillet G (1999) Whole-body positron emission tomography using ' F-fluorodeoxyglucose for post treatment evaluation in Hodgkin's disease and non-Hodgkin's lymphoma has higher diagnostic and prognostic value than classical computed tomography scan imaging. Blood, 94(2):429-433.

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Kachur AV, Dolbier WR, Jr., Evans SM, Shiue CY, Shiue GG, Skov KA, Baird IR, James BR, Li AR, Roche A and Koch CJ (1999) Synthesis of new hypoxia markers EF1 and [18F]-EF1. Appl. Rod. Isot., 51(6):643-650. Kaschten B, Stevenaert A, Sadzot B, Deprez M, Degueldre C, Del Fiore G, Luxen A and Reznik M (1998) Preoperative evaluation of 54 gliomas by PET with fluorine-18-fluorodeoxyglucose and/or carbon-11-methionine. J. Nucl. Med., 39(5):778-785. Klose T, Leidl R, Buchmann I, Brambs H and Reske SN (2000) Primary staging of lymphomas: costeffectiveness of FDG-PET versus computed tomography. Eur. J. Nucl. Med., 27(10): 1457–1464. Koh WJ, Bergman KS, Rasey JS, Peterson LM, Evans ML, Graham MM, Grierson JR, Lindsley KL, Lewellen TK and Krohn KA (1995) Evaluation of oxygenation status during fractionated radiotherapy in human non-small cell lung cancers using [F-18]fluoromisonidazole positron emission tomography. Int'l J. Rod. Onc., Bio., Physics., 33(2):391–398. Kostakoglu L and Goldsmith SJ (2000) Fluorine-18 fluorodeoxyglucose positron emission tomography in the staging and follow-up of lymphoma: is it time to shift gears? Eur. J. Nucl. Med., 27(10): 1564-1578. Kosuda S, Ichihara K, Watanabe M, Kobayashi H and Kusano S (2000) Decision-tree sensitivity analysis for costeffectiveness of chest 2-fluoro-2-D-[(18)F]fhjorodeoxyglucose positron emission tomography in patients with pulmonary nodules (non-small cell lung carcinoma) in Japan. Chest, 117(2):346–353. Lewis JS, McCarthy DW, McCarthy TJ, Fujibayashi Y and Welch MJ (1999) Evaluation of 64Cu-ATSM in vitro and in vivo in a hypoxic tumor model. J. Nucl. Med., 40(1): 177–183. Lodge MA, Lucas JD, Marsden PK, Cronin BF, ODoherty MJ and Smith MA (1999) A PET study of 18 FDG uptake in soft tissue masses. Euro. J. Nucl. Med., 26(1):22-30. Lowe VJ, Fletcher JW, Gobar L, Lawson M, Kirchner P, Valk P, Karis J, Hubner K, Delbeke D, Heiberg EV, Patz EF and Coleman RE (1998) Prospective investigation of positron emission tomography in lung nodules. J. Clin. One., 16(3): 1075–1084. Lucas JD, ODoherty MJ, Cronin BF, Marsden PK, Lodge MA, McKee PH and Smith MA (1999) Prospective evaluation of soft tissue masses and sarcomas using fluorodeoxyglucose positron emission tomography. Brit. J. Surg., 86(4):550-556. Mankoff DA, Shields AF, Link JM, Graham MM, Muzi M, Peterson LM, Eary JF and Krohn KA (1999) Kinetic analysis of 2-[HC]thymidine PET imaging studies: validation studies. J. Nucl. Med., 40(4):614-624. Mankoff DA, Shields AF, Graham MM, Link JM, Eary JF and Krohn KA (1998) Kinetic analysis of 2[carbon-ll]thymidine PET imaging studies: compartmental model and mathematical analysis. J. Nucl. Med., 39(6): 1043-1055. Minn H, Clavo AC and Wahl RL (1996) Influence of hypoxia on tracer accumulation in squamous-cell carcinoma: in vitro evaluation for PET imaging. Nucl. Med. Biol, 23(8):941–946. Okada J, Yoshikawa K, Itami M, Imaseki K, Uno K, Itami J, Kuyama J, Mikata A and Arimizu N (1992) Positron emission tomography using fluorine-18-fluorodeoxyglucose in malignant lymphoma: a comparison with proliferative activity. J. Nucl. Med., 33(3):325-329. Paquet P, Henry F, Belhocine T, Hustinx R, Najjar F, Pierard-Franchimont C, Pierard GE and Rigo P (2000) An appraisal of 18-fluorodeoxyglucose positron emission tomography for melanoma staging. Dermatology, 200(2): 167-169 [Record as supplied by publisher]. Patz EF, Jr., Lowe VJ, Hoffman JM, Paine SS, Burrowes P, Coleman RE and Goodman PC (1993) Focal pulmonary abnormalities: evaluation with F-18 fluorodeoxyglucose PET scanning. Radiology 188(2):487–490.

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Pauwels EK, Ribeiro MJ, Stoot JH, McCready VR, Bourguignon M and Maziere B (1998) FDG accumulation and tumor biology. Nucl. Med. Biol., 25(4):317–322. Rasey JS, Koh WJ, Evans ML, Peterson LM, Lewellen TK, Graham MM and Krohn KA (1996) Quantifying regional hypoxia in human tumors with positron emission tomography of [18F]fluoromisonidazole: a pretherapy study of 37 patients. Int'l J. Rad. One., Biol, Physics 36(2):417–428. Rasey JS, Koh WJ, Grierson JR, Grunbaum Z and Krohn KA (1989) Radiolabelled fluoromisonidazole as an imaging agent for tumor hypoxia. Int'l J. Rad. Onc., Bio., Physics, 17(5):985–991. Roelcke U, Radu EW, von Ammon K, Hausmann O, Maguire RP and Leenders KL (1995) Alteration of blood-brain barrier in human brain tumors: comparison of [18F]fluorodeoxyglucose, [11C]methionine and rubidium-82 using PET. J. Nemo. Sci., 132(l):20–27. Rose PG, Adler LP, Rodriguez M, Faulhaber PF, Abdul-Karim FW and Miraldi F (1999) Positron emission tomography for evaluating para-aortic nodal metastasis in locally advanced cervical cancer before surgical staging: a surgicopathologic study. J. Clin. One., 17(l):41-45. Scheidhauer K, Scharl A, Pietrzyk U, Wagner R, Gohring UJ, Schomacker K and Schicha H (1996) Qualitative [18F]FDG positron emission tomography in primary breast cancer: clinical relevance and practicability. Euro. J. Nucl. Med., 23(6):618–623. Schelling M, Avril N, Nahrig J, Kuhn W, Romer W, Saltier D, Werner M, Dose J, Janicke F, Graeff H and Schwaiger M (2000) Positron emission tomography using [18F]fluorodeoxyglucose for monitoring primary chemotherapy in breast cancer. J. Clin. Onc., 18(8): 1689–1695. Scott WJ, Shepherd J and Gambhir SS (1998) Cost-effectiveness of FDG-PET for staging non-small cell lung cancer: a decision analysis. Annals of Thoracic Surgery, 66(6): 1876–1883; discussion 1883-1885. Shields AF, Mankoff DA, Link JM, Graham MM, Eary JF, Kozawa SM, Zheng M, Lewellen B, Lewellen TK, Grierson, JR and Krohn KA (1998) Carbon-11-thymidine and FDG to measure therapy response. /. Nucl. Med., 39(10):1757-1762. Shields AF, Grierson JR, Dohmen BM, Machulla HJ, Stayanoff JC, Lawhorn-Crews JM, Obradovich JE, Muzik O and Manger TJ (1998) Imaging proliferation in vivo with [F-18]FLT and positron emission tomography. Nature Medicine, 4(11): 1334–1336. Shreve PD, Anzai Y and Wahl RL (1999) Pitfalls in oncologic diagnosis with FDG-PET imaging: physiologic and benign variants. Radiographics, 19(l):61–77; quiz 150–151. Shreve PD (1998) Focal fluorine-18 fluorodeoxyglucose accumulation in inflammatory pancreatic disease [see comments]. Eur. J. Nucl. Med., 25(3):259-264. Smith IC, Welch AE, Hutcheon AW, Miller ID, Payne S, Chilcott F, Waikar S, Whitaker T, Ah-See AK, Eremin O, Heys SD, Gilbert FJ and Sharp PF (2000) Positron emission tomography using [I8F]fluorodeoxy-D-glucose to predict the pathologic response of breast cancer to primary chemotherapy. J. Clin. Onc. 18(8): 1676–1688. Strauss LG (1996) Fluorine-18 deoxyglucose and false-positive results: a major problem in the diagnostics of oncological patients. Euro. J. Nucl. Med., 23(10):1409-1415. Sugawara Y, Braun DK, Kison PV, Russo JE, Zasadny KR and Wahl RL (1998) Rapid detection of human infections with fluorine-18 fluorodeoxyglucose and positron emission tomography: preliminary results. Euro. J. Nucl. Med., 25(9): 1238-1243. Tewson TJ (1997) Synthesis of [18F]fluoroetanidazole: a potential new tracer for imaging hypoxia. Nucl. Med. Biol. 24(8):755-760.

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Torizuka T, Zasadny KR, Recker B and Wahl RL (1998) Untreated primary lung and breast cancers: correlation between F-18 FDG kinetic rate constants and findings of in vitro studies. Radiology 207(3):767-774. Valk PE, Abella-Columna E, Haseman MK, Pounds TR, Tesar RD, Myers RW, Greiss HB and Hofer GA (1999) Whole-body PET imaging with [l8F]fluorodeoxyglucose in management of recurrent colorectal cancer. Archives of Surgery, 134(5):503-511; discussion 511–513. Vansteenkiste JF, Stroobants SG, De Leyn PR, Dupont PJ, Bogaert J, Maes A, Deneffe GJ, Nackaerts KL, Verschakelen JA, Lerut TE, Mortelmans LA and Demedts MG (1998) Lymph node staging in non-small-cell lung cancer with FDG-PET scan: a prospective study on 690 lymph node stations from 68 patients. J. Clin. Onc., 16(6):2142–2149. Wagner JD, Schauwecker D, Davidson D, Coleman JJ, 3rd, Saxman S, Hutchins G, Love C and Hayes JT (1999) Prospective study of fluorodeoxyglucose-positron emission tomography imaging of lymph node basins in melanoma patients undergoing sentinel node biopsy. J. Clin. Onc., 17(5): 1508-1515. Wahl RL, Helvie MA, Chang AE and Andersson I (1994) Detection of breast cancer in women after augmentation mammoplasty using fluorine-18-fluorodeoxyglucose-PET. J. Nucl. Med., 35(5):872-875. Warburg O, Wind F and Neglers E (1930) On the metabolism of tumors in the body. In: Metabolism of tumors. Warburg O (ed) Constable; p. 254. Warburg O (1956) On the origin of cancer cells. Science, 123:309–314. Watanabe H, Inoue T, Shinozaki T, Yanagawa T, Ahmed AR, Tomiyoshi K, Oriuchi N, Tokunaga M, Aoki J, Endo K and Takagishi K (2000) PET imaging of musculoskeletal tumours with fluorine18-methyltyrosine: comparison with fluorine-18 fluorodeoxyglucose PET. Eur. J. Nucl. Med., 27(10): 1509–1517. Weber WA, Wester HJ, Grosu AL, Herz M, Dzewas B, Feldmann HJ, Molls MK, Stocklin G and Schwaiger M (2000) 0-(2-[I8F]fluoroethyl)-L-tyrosine and L-[methyl-11C]methionine uptake in brain tumours: initial results of a comparative study. Eur. J. Nucl. Med., 27:542-549. Wester HJ, Herz M, Weber W, Heiss P, Senekowitsch-Schmidtke R, Schwaiger M and Stocklin G (1999) Synthesis and radiopharmacology of O-(2-[!8F]fluoroethyl)-L-tyrosine for tumor imaging. J. Nucl. Med., 40(1):205-212. Wurker M, Herholz K, Voges J, Pietrzyk U, Treuer H, Bauer B, Sturm V and Heiss WD (1996) Glucose consumption and methionine uptake in low-grade gliomas after iodine-125 brachytherapy. Eur. J. Nucl. Med., 23:583-586. Yamada S, Kubota K, Kubota R, Ido T and Tamahashi N (1995) High accumulation of fluorine-18fluorodeoxyglucose in turpentine-induced inflammatory tissue. J. Nucl. Med., 36(7): 1301–1306. Yang DJ, Wallace S, Cherif A, Li C, Gretzer MB, Kim EE and Podoloff DA (1995) Development of F18-labeled fluoroerythronitroimidazole as a PET agent for imaging tumor hypoxia. Radiology, 194(3):795-800. Zimny M, Bares R, Pass J, Adam G, Cremerius U, Dohmen B, Klever P, Sabri O, Schumpelick V and Buell U (1997) Fluorine-18 fluorodeoxyglucose positron emission tomography in the differential diagnosis of pancreatic carcinoma: a report of 106 cases. Euro. J. Nucl. Med., 24(6):678-682.

23. RADIOLABELED PEPTIDES FOR TUMOR IMAGING LINDA C. KNIGHT Nuclear Medicine Division, Diagnostic Imaging Department, Temple University School of Medicine, 3401 N. Broad Street, Philadelphia, PA 19140, U.S.A.

INTRODUCTION Over the last few decades, there has been an evolution in design of targeting molecules for tumor-specific radiotracer imaging. Shortly after the discovery of the methodology for producing monoclonal antibodies (MAbs), these highly specific proteins were radiolabeled and studied for the feasibility of using them for tumor imaging and therapy. It was envisioned that molecules with high binding affinity and high specificity for tumor tissue would provide excellent target-to-background contrast. This approach has proved to be far from simple, and is still undergoing modification. One difficulty with MAbs is related to their large size (approx. 145 kDa for IgG). This dictates that MAbs have a long lifespan in the blood, and thus prolonged high blood background levels. Because of their large size, MAbs may diffuse slowly into tissues to reach tumor binding sites. MAbs are primarily removed from blood by the liver, so liver background is high and this obscures hepatic metastases. Radiolabels on MAbs are mainly degraded in the liver, where the radiolabel either remains for the duration of the study, or is passed into the biliary tract and intestines, creating undesirable background in the abdomen, which makes it difficult to distinguish tumor sites from radiolabeled metabolites in the intestinal contents. Thus, antibodies generally have slow kinetics and high background. For optimal reduction of background, it is generally agreed that blood disappearance should be rapid, and removal from blood should be by the kidneys rather than by the liver. Furthermore, the kidneys should not retain the activity, but should excrete it promptly into the urine. Both the targeting molecule and the radiolabeling approach can have an effect on the biodistribution in normal tissues and the excretory route. In order to speed clearance from blood, monoclonal antibodies have been cleaved into F(ab')2 or Fab fragments, and engineered as single-chain Fvs. As size declines, the fragments have increasingly faster clearance from blood, and the smaller fragments may clear through the kidneys rather than the liver. However, the smaller fragments may also lose some of the affinity of the intact antibody, which evolved to act optimally as a larger molecule. Handbook of Radiopharmaceticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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644 Table 1. Amino Acids

Unnatural Amino Acids

Natural Amino Acids Code

Abbrev.

Name

Abbrev.

Name

A C D E F G H I K L M N P

Ala Cys Asp Glu Phe Gly His He Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr

alanine cysteine aspartic acid glutamic acid phenylalanine glycine histidine isoleucine lysine leucine methionine asparagine proline glutamine arginine serine threonine valine tryptophan tyrosine

Aba 5Ava p-Dap Hey Nal Nle pGlu Tle

aminobenzoic acid 5-aminovaleric acid ß-(L-l,2-diaminopropionic acid) homocysteine ß-naphthyl alanine norleucine pyroglutamic acid D-tert-leucine

Q R S T V W Y

It has been suggested that peptides, because of their small size and rapid blood clearance, are an attractive alternative to monoclonal antibody fragments. For the purposes of this discussion, the topic will be limited to oligopeptides of MW <10,000 Da. Peptides of this size can theoretically be filtered by the kidneys, and may clear from the blood rapidly. Many naturally-occurring peptides exhibit extremely high affinities for cellsurface receptors. Receptors for peptides are often found in higher density on tumors than in normal tissues, making it likely that radiolabeled peptide ligands could enable visualization of tumor over background. Because of their small size, peptides may be able to intercalate into tumors faster than antibodies, and be better able to reach tumor cell receptors. The length of time between radiotracer injection and diagnosis may be relatively short because of the rapid kinetics of the peptides. A potentially very important side benefit of tumor-specific peptides is their use for targeted radiotherapy. A peptide labeled with an imaging radionuclide could be used for identification of primary tumor and metastatic sites and for treatment planning dosimetry, and then the same peptide labeled with a particleemitting therapeutic radionuclide could be used for therapy. Some of the radiolabels proposed for targeted radiotherapy with peptides have included: 111in, 90Y, 114mln, 161Tb, 149Pm 90gNb and 64Cu (Anderson et al., 1995; Busse et al., 1999; de Jong et al., 1998; de Jong et al., 1995; Li et al., 2000; Tolmachev et al., 1999, Anderson et al., 1998). A few radiolabeled peptides, including [111In-DTPA]octreotide, [111in-DOTATyr3]octreotide and [90Y-DOTA-Tyr3]octreotide have already undergone initial testing in clinical trials. Amino acid abbreviations are given in Table 1.

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645

NEUROPEPTIDES Many of the peptides being studied are neuropeptides, so-called because they are primarily synthesized in the brain - see review article (Reubi, 1995b). They are also often called gut peptides, because in addition to the brain, the gut has the highest concentration of the peptides and receptors for them. The same peptides are also produced in the endocrine system (pituitary and pancreas) and are important in regulating essential biologic processes. The neuropeptides exert their action by binding to membrane-associated receptors, which are mostly G-protein coupled receptors. These consist of a complex of seven transmembrane domains, an extracellular ligand binding domain, and an intracellular domain which is linked to the activation of second messengers. After ligand binding, the ligand-receptor complex can be internalized into the cell. This is followed by lysosomal degradation of the ligand and possibly return of the receptor to the cell membrane. SOMATOSTATIN AND OCTREOTIDE Native somatostatin (somatostatin-14) is a cyclic peptide comprised of 14 amino acids. Somatostatin-28 (prosomatostatin) has an N-terminal extension of an additional 14 amino acids (see Table 2). Somatostatin plays a variety of roles, such as affecting smooth muscle contractility, endocrine and exocrine pancreatic secretion, neurotransmission and cell proliferation. There are somatostatin receptors in normal brain, anterior pituitary, endocrine and exocrine pancreas, gastrointestinal mucosa, proximal tubular epithelia of the kidneys, lymphatic tissue and vascular smooth muscle cells (Reubi, 1995b). Somatostatin receptors are expressed in high concentration in certain chronic inflammatory conditions, for example, Crohn's disease, ulcerated colitis, granulomas and rheumatoid arthritis (Reubi, 1995b). Some cells which are not part of the classical neuroendocrine system, such as lymphocytes, may also express somatostatin receptors (Krenning et al., 1993). Somatostatin receptors are also found on a number of human tumors (Krenning et al., 1993). Studies of in vitro ligand binding and autoradiographic studies with radiolabeled octreotide in specimens of human tumors have shown somatostatin receptors to be expressed on the following tumor types: gastrinoma, glucagonoma, insulinoma, carcinoid, other neuroendocrine, paraganglioma, neuroblastoma, pheochromocytoma, pituitary tumors (GH-producing), pituitary tumors (nonfunctioning), small cell lung cancer, and medullary thyroid carcinoma. Administered somatostatin is able to retard the growth of neuroendocrine tumors by interfering with growth hormone release. As an exogenously administered drug, however, natural somatostatin has a major disadvantage: it is unstable to enzymatic degradation and has a biological half-life in blood, which is too short to be useful for imaging. Octreotide (Sandostatin®) was developed as a somatostatin analogue for suppression of hypersecretion to control the symptoms of neuroendocrine diseases. The two structural features in the molecule which were found to be necessary for receptor binding are 1) the four amino acids (Phe-Trp-Lys-Thr) that form the receptor-binding domain, at the apex of the loop and 2) a cyclic conformation to constrain the four critical amino acids in an optimal configuration. Octreotide (see Table 2) contains eight amino acids, limiting the sequence to the minimal binding domain but retaining an internal disulfide crosslink to constrain the geometry of the four essential amino acids. For improved stability against enzymatic degradation in vivo, octreotide also contains D-amino acids at residues Phe1 and Trp4, and an amino alcohol, Thr(ol) in the C-

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terminal position. These structural features increased the blood half-life from 2-3 minutes for somatostatin14 to 90-120 min for octreotide. Table 2. Peptide Ligands for Somatostatin Receptors Name

Sequence

Somatostatin-14

Ala-Glv-Cvs-Lvs-Asn-Phe-Phe-Trp-Lvs-Thr-Phe-Thr-Ser-Cvs

Somatostatin-28 Octreotide

Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-LysAla-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lvs-Thr-Phe-Thr-Ser-Cvs DPhe-Cvs-Phe-DTrp-Lvs-Thr-Cvs-Thr(OL)

[Tyr3]Octreotide

DPhe-Cvs-Tyr-DTrp-Lvs-Thr-Cvs-Thr(OL)

Vapreotide

DPhe-Cys-Tyr-DTrp-Lvs-Val-Cvs-Trp-NH?

Lanreotide

DpNal-Cvs-Tvr-DTrp-Lvs-Val-Cvs-Thr-NFh

Octreotate 3

[Tyr ]Octreotate Depreotide

DPhe-Cvs-Phe-DTrp-Lys-Thr-Cvs-Thr DPhe-Cvs-Tvr-DTrp-Lys-Thr-Cvs-Thr (N-Me)Phe-Tvr-DTrp-Lvs-Val-Hcv(CH2CO-ß-Dap-Lvs-Cvs-Lvs-NH2)

NOTE: underlined portions are cyclic

It is now recognized that there are 5 Somatostatin receptor subtypes (SSTRs). Octreotide has higher affinity for Somatostatin receptor subtype 2 (SSTR2) than for SSTRS or SSTRS; it has no binding to SSTR1 or SSTR4. Somatostatin-14 and somatostatin-28 bind to all SSTRs with high affinity (Reubi, 1995b). SSTRs 14 are expressed to various extents in cancer cell lines from CNS, colon, liver, pancreas, lung, breast and skin. SSTRS is predominantly expressed in anterior pituitary, smooth muscle and GI tract (Reubi et al., 2000). It is also found in human thyroid cancer (Ain et al., 1997) and colorectal cancer (Reubi et al., 2000). In human neuroendocrine tumors, a variety of SSTR subtypes are expressed, but the most predominant subtype appears to be SSTR2 (Reubi et al., 2000), for which octreotide has high affinity. Human adenocarcinomas, such as colon cancer, have been found to express predominantly SSTR3 (Virgolini, 1997). 123

I-LABELED SOMATOSTATIN ANALOGUES Krenning et al. recognized that radiolabeled octreotide might have sufficient targeting to neuroendocrine tumors to permit imaging (Krenning et al., 1989). The initial approach was to radioiodinate the peptide, so a tyrosine-containing analogue, [Tyr3]Octreotide (TOC), was synthesized. Radiolabeling with I23 I was accomplished by direct iodination of the tyrosine side chain using chloramine-T. This radiopharmaceutical was successful in clinical imaging of several different types of tumors, including carcinoids, endocrine pancreatic tumors, meningiomas, and small-cell carcinomas of the lung (Bakker et al., 1991; Kwekkeboom et al., 1991). There were limitations with this radiopharmaceutical, however, including the inconvenience of radioiodination and purification by the end user, the relatively high cost and inadequate availability of high specific activity I23I; dehalogenation in vivo; and significant accumulation of radioactivity in liver and intestines due to hepatobiliary excretion, which hampers visualization of lesions in the abdomen.

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

647

111

IN-DTPA-OCTREOTIDE (PENTETREOTIDE)

Because of these difficulties, an

111

In-DTPA analogue of octreotide was developed. In this case, octreotide

was used as the starting molecule (with Phe in position 3 rather than Tyr as was required for the 1-123 label). DTP A was conjugated to the N-terminal DPhe1 of the peptide to facilitate labeling with radioindium. DTP A as an activated ester reacts with primary amine groups, which in octreotide exist at both the N-terminus and on the side chain of Lys5. Since Lys5 is in the binding region of the molecule, modification at this residue must be avoided. Therefore, the side chain of Lys5 must be blocked during conjugation of DTPA. This can be accomplished by using a removable protecting group (Boc) for protection of the Lys sidechain. After DTPA conjugation, the Boc group can be removed by trifluoroacetic acid (TFA) treatment. Labeling DTPAoctreotide is carried out by adding ln ln in an acetate-buffered medium and labeling is nearly quantitative within 30 minutes at room temperature. In vivo, 111In-DTPA-octreotide has primarily renal excretion, with very little hepatobiliary excretion. This feature allows clear visualization of abdominal tumor sites. Although it has a slower initial rate of clearance from blood, at 24 hr it has lower background levels than I23I-TOC, because 111 In-DTPA-octreotide has lower circulating levels of degradation products (Krenning et al., 1993). Recommended imaging times are at 4 and 24 hours post injection. The additional background clearance between 4 and 24 hours results in better visualization of small lesions. lllIn-DTPA-octreotide in plasma in vivo is mainly in its original peptidebound form through 4 hr. Degradation in plasma samples was observed only in samples drawn at later times, when the level of circulating activity was less than 10% of the administered dose (Krenning et al., 1992). 111 In-DTPA-octreotide appears to be excreted intact in urine. The long residence time in kidneys, however, suggests that following glomerular filtration, some of the labeled peptide is reabsorbed by the tubules. Normal tissue distribution by 24 hr includes uptake in pituitary, thyroid, liver, spleen, kidneys, and urinary bladder. Accumulation in nasal region and lung hili may be seen in common cold/influenza; this is thought to be related to binding to activated lymphocytes (Krenning et al., 1992). In patients who are receiving octreotide treatment there may be lower uptake of 111in-DTPA-octreotide in the spleen because of saturation of receptors by unlabeled ligand (Krenning et al., 1992). After somatostatin binds to SSTR2, the ligand/receptor is internalized into the cell (Andersson et al., 1996; Hofland et al. 1995). This can be very advantageous for radionuclide targeting, because it improves ligand retention over surface binding, which is potentially reversible. Andersson et al. showed that 111In-DTPAoctreotide was internalized into cultured gastric carcinoid, midgut carcinoid and glucagonoma cells. Electron microscopic studies revealed that most of the internalized 111ln was located in the cytoplasm of the tumor cells, although a small fraction appeared to be in the cell nucleus. Localization in the cell nucleus would be advantageous for optimal therapeutic use of 111 In-DTP A-octreotide, because of the short range of 111In Auger electrons (Andersson et al., 1996). Renal uptake and retention of radioactivity is quite prominent following administration of

111

in-DTPA-

octreotide. This may be a problem for visualization of tumors near the kidneys, and may be dose-limiting for therapeutic uses of radiolabeled octreotide. It is believed that 111In-DTP A-octreotide, like most small peptides, is excreted by glomerular filtration, but is then efficiently reabsorbed in the proximal tubules. The

648

HANDBOOK OF RADIOPHARMACEUTICALS

reabsorption process involves binding to a carrier on the tubular cell membrane, internalization in an intracellular vesicle, release of the vesicular contents to lysosomes, followed by degradation in the lysosome (de Jong el al., 1996). The primary labeled metabolites become trapped in the lysosomes of the proximal renal tubular cells (Duncan et al., 1997). A simple way to prevent the renal reuptake is being sought. Because membranes of renal tubular cells contain negatively charged sites which favor binding of positively charged molecules, changing urine pH can affect reabsorption of '"in-DTPA-octreotide. In rats, altering urine pH by agents administered in food induced decreases in renal retention of 111In-DTPA-octreotide (de Jong et al., 1996). It has been shown that co-administration of positively charged amino acids such as lysine can inhibit reabsorption of radiolabeled peptide by blocking the renal reuptake mechanism. In rats, intravenous injection of 400mg/kg lysine immediately before 111In-DTPA-octreotide reduced tumor uptake 40% compared to controls (de Jong et al., 1996). In initial studies in humans, infusion of an amino acid mixture including lysine and arginine reduced kidney retention by about 50% compared to controls (Hammond et al., 1993). The most common metabolites of 111In-DTPA-octreotide which are extractable from liver, kidney, tumor, pancreas, and blood appear to be 111'in-DTPA-Phe-Cys-OH and 111in-DTPA-Phe-OH. With time, more of the latter is formed, suggesting that the peptide is sequentially degraded in vivo. By 24 hr post injection, (when imaging with 111In-DTPA-octreotide is usually carried out), most of the activity appears to be metabolites rather than intact labeled octreotide (Bass et al., 1998). Intracellular metabolites are apparently trapped because there is no efficient transport system to remove them from cells; hence, alternate approaches to attaching metal chelators could aid in clearance of radiolabels from tissues. It was initially hypothesized that the highest specific activity 111In-DTPA-octreotide would give the best receptor binding and therefore the best images in vivo. Recent data have suggested that this is not the case. In normal rats, administration of small amounts (0.5 - 5 ug/rat) of cold (unlabeled) octreotide shortly before, during, or shortly after injection of radiotracer actually improved uptake of 111 In-DTPA-octreotide in SSTR rich tissues (Breeman et al., 1995). It was suggested that cold octreotide may have effects on competition with endogenous somatostatin in somatostatin-producing tissues, exposure time of the receptor to the radiopharmaceutical, or rate of internalization of the receptor-ligand complex, and the rate of re-expression or upregulation of the receptor. The last two hypotheses are supported by studies in vitro. Increased internalization of 123I-octreotide into tumor cells in vitro was observed in the presence of small amounts of cold octreotide (Hofland et al., 1995). In other studies, treatment of pituitary cells with somatostatin caused an increase in the number of somatostatin receptors exposed on the cells (Presky & Schonbrunn, 1988). 111

In-DTPA-octreotide has been extensively studied in routine clinical use. It has high diagnostic capability for detecting neuroendocrine tumors and lymphomas. The tumors that are detected well by DTPAoctreotide, endocrine tumors of the digestive tract, are uncommon, accounting for approximately 2% of all malignant gastrointestinal neoplasms (approx. 0.7 cases/100,000/yr) (Oberg et al., 19%). The sensitivity of 111 In-DTPA-octreotide for other tumors is lower. Sensitivity for medullary thyroid cancer is 47-68% (Seregni et al., 1998). A number of more frequently occurring cancers do not express SSTR2, and so they

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

649

cannot be imaged with octreotide. An improved agent for detection of more common cancers is therefore sought. At the same time, other radiolabels are being pursued for octreotide, to optimize its use for imaging and therapy, 99M

TC LABELED OCTREOTIDE ANALOGUES

The rapid blood clearance and fast tumor uptake of octreotide raised the idea that a Tc label might be suitable. Several labeling approaches have been proposed: Direct Labeling Approach. This approach involves intentional reduction of octreotide's internal disulfide bond to create two free thiols which are used to complex reduced Tc-99m, and theoretically retain a cyclic configuration through the coordination of Tc. In the first reported attempt with this method, a mixture of two products resulted, which did not show selective uptake in SSTR-rich tissues in vivo (Kolan et al., 1996). With the aid of molecular modeling, Ferryman et al. designed octreotide analogs containing the metalbinding sequence Cys-Cys-Xn-Cys for complexing reduced Tc. The three Cys thiols and the second Cys's amide nitrogen are used to form the metal coordination sphere. This enables cyclization of the peptide through Tc or Re to avoid a pendant chelating group and maintain small size for optimal diffusion into tumors (Ferryman et al., 1999). Bifunctional Chelating Agent Approach. Maina et al. coupled PnAO to [Fmoc-Lys5]octreotide by reacting an isothiocyanate derivative of PnAO with the amino terminus to result in a thiourea linkage. After deprotection and conjugation, the conjugate was labeled by ligand exchange from

99m

Tc-tartrate at room

temperature. The labeling required large quantities of cold peptide in order to achieve reasonable labeling efficiency, followed by HPLC purification to remove excess cold peptide. HPLC-purified material was tested for uptake in pancreatic tumors and other somatostatin-receptor rich tissues in rats. Tumor uptake was ().38%!D/g at 90 min. Unfortunately, the lipophilic character of the complex caused it to have considerable hepatobiliary uptake (liver = 4.5% ID/g at 90 min) and long blood residence time (blood - 0.65 %ID/g at 90 rnin). The investigators concluded that the high liver uptake rendered the agent unsuitable for detecting tumors in the abdomen (Maina et al., 1994). Preformed Chelate Approach. Spradau et al. labeled octreotide with cyclopentadienyltricarbonyltechnetium (99mTc-CpTT). In a novel double ligand transfer reaction, pertechnetate was reduced and functionalized to produce Tc-CpTT methyl ester intermediate. This was transformed into a HABt-activated ester, which was coupled to [Boc-Lys5]octreotide.

After purification, the Boc protecting group was removed with TFA,

yielding the final product in 8% overall yield. The production of the preformed chelate and the coupling are at present very complex, but the resulting labeled peptide was prepared in very high specific activity. The label was stable in human plasma at 37°C. In normal rats, the labeled peptide was specifically taken up in SSTR-rich tissues (adrenals and pancreas). This conjugate was excessively lipophilic, however, and exhibited significant hepatobiliary excretion (Spradau et al., 1999).

650

HANDBOOK OF RADIOPHARMACEUTICALS

LABELING OCTREOTTOE FOR PET Octreotide labeled with a positron-emitter would enable imaging with PET to provide quantitative assessment of tracer deposition in tissues. This would be an advance over SPECT imaging with octreotide in which tissue uptake is only semiquantitative. Thus, PET could be helpful in quantifying the number of somatostatin receptors and in predicting clinical response (Anderson et al., 1995). Octreotide has been labeled with 67Ga/68Ga using desferrioxamine (DFO) as a chelator (Smith-Jones et al., 1994). Preparation of this radiopharmaceutical involves several steps; however, it is amenable to formulation of a kit for labeling in the final step. [Boc-Lys5]octreotide was reacted with succinic anhydride to place a succinate group on the N-terminus. After purification, this was reacted with the free base form of DFO in the presence of dicyclohexyl carbodiimide (DCCI) and hydroxybenzotriazol (HOBt). The product was treated with TFA to deprotect the sidechain of Lys5. Labeling was accomplished by combining 67/68Ga in ammonium acetate buffer with DFO-octreotide in dilute acetic acid. Labeling with no-carrier-added 67Ga was >99.5% after 20 min at room temp at pH 5.5, and increased with further incubation. Labeling with 68Ga required dissociation of the generator eluate complex (68Ga-EDTA). Chelation of 68Ga by DFO-octreotide (at pH 4.5) was optimal (98%) after 5 min and decreased with further incubation, possibly because of radiolysis effects. Good stability in serum suggested that the relatively large DFO attached to octreotide prevented 67Ga transchelation to serum proteins (3.5% in 24 hr) better than DTPA-octreotide (15.7% in 24 hr). In vitro the receptor binding affinities of DFO-octreotide and Ga-DFO-octreotide were only slightly decreased from unmodified octreotide, indicating that the bulky chelator is tolerated by the relatively small peptide if it is optimally placed away from the binding domain. Blood clearance in tumor-bearing rats was rapid and islet cell tumors were clearly visible shortly after injection with either planar (67Ga) or PET (67Ga) imaging. Region-ofinterest analysis of PET data indicated that maximal tumor uptake occurred at about 25 min, with a gradual washout thereafter. Tumor uptake at 1 hr was 0.38%ID/g, with tumor-to-blood of 2.5 and tumor-to-muscle of 7.4. Excretion was predominantly renal. There have been several attempts to label octreotide analogues with 18F for PET. Since direct fluorination of peptides is difficult, Guhlke et al. reacted [18F]-fluoropropionic acid 4-nitrophenyl ester with the N-terminus of [Boc-Lys5]octreotide (Guhlke et al., 1994). After acylation, the Lys sidechain was deprotected by treatment with TFA. HPLC purification produced pure product in 65% radiochemical yield. The binding affinity of l9F-labeled octreotide was similar to that of [nat!n-DTPA]octreotide, when measured in competition with I25I-SDZ 204-090. Hosteller et al. used a 4-[l8F]fluorobenzoyl moiety, which was attached to the N-terminus of [Boc-Lys5]octreotide using DCCI, followed by deprotection of Lys5. Coupling yields were variable (8-55%). In tumor-bearing rats, the radiotracer appeared to be somewhat lipophilic, as there was appreciable liver uptake. There was still modest uptake in tumor (0.28 %ID/g at 1 hr) and SSTR-rich organs (adrenals = 1.09 %ID/g and pancreas = 0.28 %ID/g), which were mostly blockable by excess cold octreotide (Hosteller et al., 1999). The physical half-lives of F (110 min) and Ga (68 min) may be too short for optimal imaging of octreotide because of the need to wait for background clearance ('"in-DTPA-octreotide shows optimal

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

651

tumor: background ratios at 24 hr). Therefore, a longer-lived positron emitter may be needed for labeling octreotide. Anderson et al. have proposed 64Cu (T \a =12.8 hr) as an alternative metal label for octreotide (Anderson et ai, 1995), In order to complex copper, the chelators TETA and CPTA were conjugated to the N-terminus of [Boc-Lys5]octreotide.

Labeling of the peptide conjugates with copper-64-acetate was carried out in

ammonium acetate buffer, pH 5.5, at room temperature. Incorporation of >95% of 64Cu was achieved with TETA-octreotide within 60 min. Incorporation into CPTA was slower, reaching a maximum of 85% radiochemical purity after 12–18hr. Addition of gentisic acid (1 mg/ml) was helpful to protect the conjugates against radiolysis. The 64Cu-octreotide conjugates had binding affinities 10-40x as high as 111InDTPA-octreotide for AtT20 mouse pituitary carcinoma cell membranes in vitro. In rats, 64Cu-CPTAoctreotide exhibited high liver uptake, however 4Cu-TETA-octreotide had low liver uptake (comparable to 111 In-DTPA-octreotide). In addition, the kidney clearance of 64Cu-TETA-octreotide was faster than that of 111 In-DTPA-octreotide (Anderson et al., 1995). In clinical trials, more tumors were identified with 64CuTETA-octreotide and PET than with 111In-DTPA-octreotide and conventional scintigraphy (Lewis et al., 1999b). OTHER SOMATOSTATIN ANALOGUES [Tyr3]Octreotide [Tyr3]octreotide (TOC) was developed in order to create an octreotide analogue with a site for radioiodination. However, TOC may have an advantage over octreotide in that substitution of Phe3 with Tyr3 increases hydrophilicity. Instead of labeling it with radioiodine, TOC has been modified at the Nterminus with bifunctional chelators such as DTPA to coordinate radiometals. Compared with 111In-DTPAoctreotide, 111In-DTPA-TOC had similar binding to cell membranes in vitro, but had higher internalization in vitro and higher tumor uptake in rats. Both 111in-labeled octreotide analogues were stable in vivo, and were excreted essentially undegraded in urine (de Jong et al., 1998). In order to more stably complex other metals, especially Yttrium, DOTA has been coupled to TOC. Compared with (unlabeled) DTPA-octreotide, (unlabeled) DOTA-TOC had 5-fold higher potency for inhibiting 125I-TOC binding to mouse AtT20 pituitary cell membranes (de Jong et al., 1998). [Tyr3]oetreotate (Y3-TATE) Y3-TATE differs from TOC in that the C-terminal Trp bears the conventional carboxylic acid instead of octreotide's alcohol. This modification provides an additional negative charge on the C-terminus compared to Tyr3-octreotide. 111In-DTPA-Y3-TATE was comparable to 111In-DTPA-octreotide for binding to cell membranes in vitro, but had dramatically higher internalization and higher binding to tumors in vivo, especially at 4 hr post injection (de Jong et al., 1998). In humans, 111ln-DTPA-Y3-TATE cleared rapidly from blood, with 3.2% ID remaining in blood at 10 min, and 0.3% at 24 hr. Essentially intact labeled peptide was found in blood and urine samples taken at 1 hr post injection (Bakker et al., 2000). In more recent studies, deJong et al. reported that DOTA-Y3-TATE had a higher uptake and internalization into cells in

652

HANDBOOK OF RADIOPHARMACEUTICALS

vitro when labeled with I25I rather than 111ln. Thus, "empty" DOTA (not filled with binding and uptake (De Jong el al., 2000a).

111

ln) enhanced the

Y3-TATE has also been conjugated to TETA for labeling with 64Cu. 64Cu-TETA-YS-TATE exhibited a Kd of 549 pM in CA20948 cell membranes, compared with a Kd of 617 pM for 64Cu-TETA-OC. In rats bearing CA20948 pancreatic tumors, tumor uptake of 64Cu-TETA-YS-TATE was 2.37% ID/g at 1 hr, compared with 1.09 %ID/g for 64Cu-TETA-OC. In mice bearing AR42J pancreatic tumors, tumor uptake of 64Cu-TETAY3-TATE was 21.6 % ID/g at 1 hr, compared with 11.24 %ID/g for 64Cu-TETA-OC. Receptor-mediated uptake in pancreas, pituitary, adrenal glands, bone and lungs were higher for 64Cu-TETA-YS-TATE. When compared with other octreotide analogues, uptake in AR42J pancreatic tumor cells in vitro was higher for 64 Cu-TETA-Y3-TATE (61%) vs. 64Cu-TETA-TATE (56%) than for 64Cu-TETA-TOC (47%) or 64Cu-TETAoctreotide (34%), suggesting that the C-terminal carboxylate contributes more to receptor binding and internalization than Tyr substitution at the 3-position (Lewis et al., 1999a). A new tricarbonyltechnetium complex, fac[99mTc(CO)3(H2)3]+, may be used to label histidine in peptide chains, without risk of reduction of disulfide bridges (Waibel et al., 1999). This technology was applied to an octreotate analogue, [His°,Tyr3]octreotate, containing an N-terminal His to act as a labeling site. High specific activity was achieved, 7000 Ci/mmol, and the label was stable for 24 hr. In rats, the labeled peptide showed uptake in SSTR expressing tissues in pancreas, adrenals and pancreatic tumor. However, uptake in the liver and GI tract indicated unwanted lipophilicity (Marmion et al., 1999). This may be partly due to plasma protein binding, as N-terminal His residues are only bidentate in their coordination for Tc-tricarbonyl, leaving an opportunity for plasma protein to fill a coordination site (Schubiger et al., 1999). A tridentate His analogue can be created by using Na-His-Ac in place of histidine on the N-terminus. [99mTc-(CO)3-(Na-HisAc)°,Tyr3]octreotate had better tumor background ratios at 4 hr post injection in rats bearing CA20948 tumor than other bifunctional chelating agents (DTPA, EDTA, IDA, NOTA, DOTA, His) placed on N-terminus. The overall charge of the peptide appeared to be important also, with the more negatively charged peptides (charge less than -2) having more favorable biodistribution than more positively charged peptides (Marmion et al., 2000). GLYCATED OCTREOTIDE ANALOGUES Peptides with lipophilic character display unwanted hepatobiliary excretion. Leisner et al. attempted to reduce the lipophilicity of radioiodinated and radiofluorinated octreotide analogues by introducing carbohydrate moieties onto the N-terminus of TOC. After synthesis and cyclization of [Boc-Lys5]TOC, the peptide was modified at the N-terminus to create 1-deoxy-D-fructose, D-maltose, or D-maltotriose derivatives. These were radioiodinated at the Tyr3 position by direct (lodogen®) radioiodination. All three glycosylated analogues exhibited lower liver uptake and slower blood clearance than 125I-TOC. One derivative, N-(a-D-glucosyl(l-4)-l-deoxy-D-fructosyl)-[125I]-TOC, exhibited liver uptake that was onetenth that of the unglycosylated tracer (Leisner et al. 1999). In tumor-bearing mice, I25l-glycated TOC had lower liver uptake than I25I-TOC (1.0 - 2.7% ID/g vs. 3.2 %ID/g, at 1 hr) (Schottelius et al, 2000). Glycosylation did not affect the ability of the peptides to accumulate in SSTR-rich tissues such as pancreas and adrenals, confirming that it is possible to modify Phe1 without affecting receptor binding (Leisner et al..

RADIOLABELED PEPTIDES FOR TUMOR IMAGING 1999).

653

In further studies, glycated derivatives of Y3-TATE were also prepared and labeled with

Glycated

125

I-Y3-TATE lowered the liver levels further to 0.5-0.8 %ID/g, compared with unmodified

Y3-TATE (liver - 1.2 %ID/g). Tumor uptake was also improved for glycated compared with unmodified

!25 I25

I.

I-

I25

I-Y3-TATE (13-25 %ID/g)

125

I-TOC (5.8 %ID/g) (Schottelius et al., 2000).

VAPREOTIDE (RC-160) This analogue of octreotide differs from TOC in that it has Val instead of Thr6, and Trp8 instead of Thr8(OL) (see Table 2). Vapreotide was found to have enhanced binding affinity to SSTR4 when compared to octreotide (Schally, 1988). This raised the hopes that it would recognize a broader spectrum of tumor types than octreotide, although vapreotide's affinity for SSTR4 is much lower than for SSTR2 (Reubi et al,, 2000). It is alleged that vapreotide can cross the blood-brain barrier. Although some have speculated that this might enable better targeting of brain tumors, this property was noted for unlabeled peptide. It is unlikely that after modification with metal chelates vapreotide would be sufficiently lipophilic to cross the blood-brain barrier, nor would it be desirable for it to be that lipophilic for imaging in the rest of the body. Bifunctional chelating agents were coupled to the N-terminus for labeling with radiometals. Decristoforo and Mather compared TOC and vapreotide labeled with several different Tc chelating cores, including benzoyl-MAG3 and N3S-adipate, ETDA, Bz2EDTA, and HYNIC with various coligand systems, including tricine, tricine+nicotinic acid and EDDA (Decristoforo & Mather, 1999a-c). In vitro, all of the peptide conjugates retained the binding affinity of the unmodified peptides. When these conjugates were labeled with 99mTc, they all showed high specific binding to AR42J membranes. Internalization studies were performed in live AR42J cells. Tc-MAG3 -vapreotide, Tc-N3S-vapreotide, and Tc-HYNIC-vapreotide (with various coligands) all had high nonspecific binding to cells which made quantification of internalization difficult. Internalization was slower for Tc-EDDA/HYNIC-vapreotide compared with Tc-EDDA/HYNIC-TOC (Decristoforo & Mather, 1999c). The labeled peptide conjugates differed markedly in their tumor uptakes and biodistribution in rats. When labeled with the same core and coligand, TOC had higher tumor uptake in vivo than vapreotide (Decristoforo & Mather, 1999b). The most hydrophilic complexes (using EDDA as coligand with HYNIC) had the most favorable biodistribution, with the lowest liver uptake and the fastest blood clearance. Unfortunately, this labeling approach was hampered by a 64-69% labeling yield, whereas the other labeling approaches achieved nearly quantitative labeling. The MAG3 and N3S conjugates had extensive hepatobiliary excretion.

For a

99m

given chelating agent, there was more plasma protein binding with Tc-vapreotide than with Tc-TOC (Decristoforo, 1999b). 99mTc-vapreotides were more hydrophobic, as judged by later retention times on RPHPLC (Decristoforo & Mather, 1999b). Even with a single peptide sequence and position of chelator, apparently small factors can have a large effect on biodistribution. For HYNIC-vapreotide, when EDDA was used as coligand, there was primarily renal excretion with hardly any liver excretion. With tricine as coligand, there was combined renal and hepatic clearance. With lipophilic substituted coligands such as Bz2EDDA, there was predominantly liver uptake,

654

HANDBOOK OF RADIOPHARMACEUTICALS

and plasma protein binding was also noted. Ternary ligand systems, such as tricine with nicotinic acid or tricine with 3,5-pyridinedicarboxylic acid, exhibited reduced protein binding in vitro compared to tricine alone, and reduced liver and blood levels in vivo (Decristoforo & Mather, 1999a). LANREOTIDE Lanreotide (Somatuline®; LAN) differs from TOC in that it has 3-[2-naphthyl]-alanine at the N-terminus in place of DPhe as in octreotide, Val6 instead of Thr6 and Thr-NH2 at the C-terminus instead of Thr-Ol. DOTA-lanreotide (also known as MAURITIUS) has receptor binding behavior which is different from unmodified lanreotide and all other known somatostatin analogues, in that it binds to all SSTRs 2-5 with high affinity (Kd 1-10 nM) (Virgolini et al. 1998c). SSTR3 is found in many human cancers including adenocarcinomas, but only moderately binds ['"in-DTPA-DPhe'loctreotide. '"in-DOTA-lanreotide exhibited high binding in vitro to a wide range of human primary tumor specimens, including intestinal adenocarcinomas and breast cancer, as well as to cells from several breast, prostate, colon and pancreatic cancer cell lines (Smith-Jones et al., 1998). In clinical studies, Virgolini et al. found that 111In-DOTA-lanreotide was tolerated well without apparent side effects (dose of peptide - 7 nmol). Blood disappearance of the labeled peptide was rapid, with < 3% remaining in the blood at 2 hr. Urine excretion was 21% and 42% ID by 4 and 24 hr, respectively. Up to 24 hr, the form of radioactivity in urine was primarily intact peptide. Imaging (both planar and SPECT) was carried out at 4-6 hr or 24 hr post injection, although sites of tumor uptake were still visible at 72 hr post injection. A variety of known primary and metastatic tumors were visualized, including carcinoid, gastrinoma, pheochromocytoma, lymphoma, pancreatic adenocarcinoma and colorectal adenocarcinoma. In comparison with 111in-DTPA-octreotide in a subset of the same patients, 111In-DOTA-lanreotide had slower whole-body clearance, lower kidney retention, and higher estimated uptake in tumors. Liver uptake of 111InDOTA-lanreotide was about 40% higher than 111In-DTPA-octreotide (Virgolini et al., 1998c). DEPREOTIDE Because of difficulties in accommodating a thiol-containing Tc chelator, and the risks associated with performing Tc reduction in the presence of a disulfide bond, Pearson et al. synthesized an octreotide analogue, depreotide (P829) (Table 2). Depreotide is comprised of a cyclic hexapeptide (containing the pharmacophore Tyr-DTrp-Lys-Val), which is cyclized by an amide bond between Phe1 and Hey6. This is linked through the thiol group of Hey to a linear tetrapeptide containing a sequence for complexing reduced Tc: p-Dap-Lys-Cys, where (3-Dap = p-(L-l,2-diamino propionic acid). This peptide sequence forms a monoamine, bisamide, monothiol chelator for Tc(V) (Vallabhajosula et al., 1996). Technetium-99m complexes were prepared by ligand exchange from ""Tc-glucoheptonate. The oxorhenium complex of P829 had 20-30x higher affinity for AR42J cells than the unmetallated peptide (Pearson et al., 19%). P829 appears to have a wider range of SSTR recognition than 111in-DTPA-octreotide. It appears to recognize SSTR3, which is found in many human cancers but only moderately binds [111in-DTPA-DPhe'joctreotide (Virgolini, 1997). It also appears to recognize SSTR2 and SSTRS, but with slightly lower affinity than [111In-DTPA]octreotide (Virgolini et al., 1998b).

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

655

In rats bearing rat pancreatic tumors, tumor uptake of 99mTc-P829 was slightly higher than 111In-DTPAoctreoiide (4.8 vs. 2.8 %ID/g at 90 min). Tumonblood (21 vs. 29) and tumonmuscle ratios (68 vs. 90) were comparable between the two tracers. Less than 5% was found in the GI tract at 1 hour, with 30% in the kidneys and 20% in urine (Vallabhajosula et al, 1996). In human subjects, P829 was able to detect sites of breast cancer and melanoma (Virgolini et al., 1998b). In contrast, another peptide (P587) evaluated by the same group had rather different biodistribution despite its structural similarity. This peptide contained the same receptor-binding peptide core, but the appendage for Tc coordination was Gly-Gly-Cys-Lys-NH2. This peptide had approximately the same tumor uptake (4% ID/g at 1.5 hr), but exhibited about 40% gastrointestinal uptake by 1 hr post injection, with 25% urinary bladder and 6% in kidneys. It was found that the relative positions of charged amino acids in the chelator sequence affected receptor binding affinity as well as general biodistribution (Pearson et al., 1996). VASOACTIVE INTESTINAL PEPTIDE Vasoactive intestinal peptide (VIP) is a neuropeptide of 28 amino acids, with no internal disulfide crosslinks (see Table 3). VIP is closely related to pituitary adenylate cyclase activating polypeptide (PACAP). Both belong to a group of ligands for a G-protein coupled receptor subfamily which includes receptors for secretin, glucagon, glucagon-like peptide, VIP, PACAP, growth hormone-releasing factor (GRF), gastric inhibitory peptide, corticotrophin releasing factor, parathyroid hormone, and calcitonin (Cao et al., 1998). There are two known VIP receptors (VIPR). VIPR1 is found mainly in the CNS and has higher affinity for PACAP than for VIP. VIPR2 is found in peripheral tissues and binds VIP and PACAP equally (Cao et al., 1998). Secretin and GRF have only weak affinity for VIP/PACAP receptors. Table 3. Peptide Ligands for VIP Receptors Name

Sequence

VIP

His-Ser- Asp-Ala- Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-GlnMet-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-GlnMet-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Aba-Gly-Gly-DAla-Gly His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-GlnHcy(CH2CO.Gly-Gly-Cys-Lys-NH2)-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 3

, Arg" ]VIP His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-GlnMet-Ala-Val-Lys-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn

PACAP (1 -27)

His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-GlnMet-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu

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VIP has a variety of biologic activities, including vasodilation, stimulation of the secretion of various hormones, modulation of immune response, and stimulation of the growth and proliferation of normal and malignant cells - see review article (Virgolini et al., 1995). These actions are mediated by VIP binding to cell surface receptors, which are found in many tissues, including lungs, GI tract, kidneys, and peripheral blood cells. Of interest for oncology, many tumors express VIP receptors in higher concentrations than in surrounding normal tissues or blood cells, permitting detection of these tumors by radiolabeled VIP scintigraphy (Virgolini et al., 1995). In vitro, [125I-Tyr10]VIP bound to receptors on tissue sections of a wide range of cancer types, including all specimens tested of breast carcinomas, breast carcinoma metastases, prostate carcinoma and prostate carcinoma metastases, bladder carcinoma, endometrial carcinoma, colon adenocarcinoma, gastrointestinal squamous cell carcinomas, lymphomas, meningiomas and astrocytomas.

Most ovarian adenocarcinoma,

pancreatic adenocarcinoma, non-small-cell lung cancer, and glioblastoma were receptor positive (Reubi, 1995a). Virgolini et al. reported that many tumors co-express receptors for VIP and somatostatin (Virgolini et al., 1995). Subsequently, studies found that the receptor recognized by VIP is probably SSTR3 (Virgolini et al., 1998a). VIP binds avidly to SSTR3, in contrast to octreotide, which preferentially recognizes SSTR2 (Virgolini, 1997). Thus, scintigraphy with radiolabeled VIP may have advantages over [111in-DTPAJoctreotide in that it recognizes a wider variety of tumors, including some of the more common and devastating cancers. The active site of the VIP molecule may not be a contiguous range of amino acids. The molecule is disordered in aqueous solution; however, residues 9-17 and 23-28 assume helical conformations under conditions which simulate the vicinity of a receptor (Wray et al., 1998). It is probable that a helical domain interacts with the receptor. The molecule cannot be shortened at either end without loss of activity. The peptide contains two tyrosines at positions 10 and 22, and either or both may be labeled by direct radioiodination. [125I-Tyr10,Tyr22JVIP prepared by direct iodination has been shown to be active and has been used extensively as a ligand to study the VIP receptors in vitro (Virgolini et al., 1995). For imaging studies, naturally occurring VIP has been labeled with 123I by the lodogen® method (Virgolini et al., 1995). After labeling, purification by HPLC was performed to separate labeled from unlabeled peptide. Two radioactive peaks corresponding to [123I-Tyr'°]VIP and [l23I-Tyr22]VIP were obtained, and were pooled together for in vivo studies. I23

I-VIP bound to membranes from adenocarcinoma tumor cells and cell lines (COLO321, HT29) in vitro.

The observed dissociation constants were comparable to Kds for VIP binding to membranes of platelets and monocytes, but with 150-10,000x higher receptor density on the cancer cell membranes (Raderer et al., 1996). 123I-VIP recognizes approx. l000x as many binding sites per VEPoma cell as it recognizes per cell on peripheral monocytes and the affinity for VIPoma cells is approximately 3x as high (Virgolini et al., 1998a). For human studies, the high potency of VIP for inducing vasodilation required that the highest possible specific activity be prepared. Virgolini et al. purified I23 I-VIP by HPLC to remove unlabeled peptide. When

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300 pmol (< 1 pg) VIP was administered, human subjects experienced a transient drop in blood pressure during the first minutes after administration (Virgolini et al., 1995). VIP has a short half-life in the blood. Unlabeled VIP has a T1/2 in canine circulation of less than 3 minutes, as measured by immunoreactivity (Kabemura, 1992). I23I-VIP had a blood Tj/2 of about 1 minute in human subjects (Virgolini et al., 1995). Although the metabolites have not been explicitly determined, it is probable that the molecule is rapidly broken down in vivo. As with other natural linear polypeptides, VIP is susceptible to proteolytic degradation (Mather, 2000). In human subjects, 123I-VIP had pronounced uptake in the lungs, representing about 40% of the injected dose. No other substantial organ accumulation was noted. Urinary excretion in humans by 4, 8 and 24 hours amounted to 37, 68 and 93% of the administered dose (Virgolini et al, 1995). 123I-VIP has been able to image a number of human cancers, including adenocarcinomas, breast cancers, neuroblastomas, melanomas, and pancreatic carcinomas. Interestingly, sensitivity is higher (90%) for smaller pancreatic tumors than for larger ones (32% sensitivity), possibly because of changes in receptor expression, or because of tumor changes that affect radiotracer delivery (Raderer et al, 1998). Both 123I-VIP and 123In-DTPA-octreotide are able to image primary and metastatic carcinoid tumors, although 123In-DTPA-octreotide has higher sensitivity for these types of tumors (Virgolini, 1997). l!1 InDTPA-octreotide is also more sensitive for lymphomas and breast cancer. Pulmonary lesions cannot be visualized with 123I-VIP because of normal lung uptake (approx. 40% ID) (Raderer et al, 2000). 123I-VIP scintigraphy has a low sensitivity for other endocrine tumors, such as gastrinomas, pheochromocytomas, unclassified neuroendocrine tumors, glucagonomas and medullary thyroid cancer. In contrast, [ 111 n-DTPADPhe1]octreotide is more efficacious for neuroendocrine tumors, but not useful for imaging gastric, colorectal or pancreatic adenocarcinomas. In order to improve detection of occult cancers, a "cocktail" scan has been proposed in which both of these radiopharmaceuticals are given together and imaging for both 123I and 111ln is carried out. This technique unfortunately sacrifices some of the advantages of performing the !23 I-VIP study alone, such as the favorable biodistribution with minimal background except for lungs, and the possibility of using lower energy, higher resolution collimation for 123I. In order to label VIP with 99mTc, Kolan et al. attached bifunctional chelating agents, including MAG3, HYNIC and CPTA to VIP primary amino groups (Kolan et al, 1997). The Tc-labeled products had poor tumor uptake in mice. It was concluded that modification of the N-terminal His was responsible for loss of activity; however, three midchain lysines were not blocked prior to modification and could have been unintentionally modified. The possibility of midchain modification is further substantiated by radio-HPLC showing multiple peaks (Pallela et al., 1999). Subsequently, the same group prepared an analogue of VIP which had been extended at the C-terminus during solid phase peptide synthesis to include aminobenzoic acid-Gly-Gly-DAla-Gly (Pallela et al., 1999). This additional sequence acts as a spacer followed by a tetrapepdde sequence designed to complex reduced Tc, This analogue, named TP3654, retained functional activity in an in vitro assay (Pallela et al., 1999). In

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nude mice bearing human colon tumor xenografts, tumor uptake of 99mTc-TP3654 at 4 hr was much lower than that of 125I-VIP (0.24 vs. 2.22%ID/g), but its metabolites were retained better by tumor. At 24 hr tumor levels of 99mTc-TP3654 were higher than that of 125I-VIP (0.23 vs. 0.06 %ID/g). Unfortunately,99mTois not an optimal nuclide for imaging at 24 hr. Although there was uptake in the lungs which could be blocked by administration of unlabeled peptide, the lung uptake of 99mTc-TP3654 (0.17%ID/g at 4h) was lower than expected, and much lower than that of I23I-VIP (3.98%ID/g at 4h) (Pallela et al., 1999). In an initial clinical trial of 11 patients, 99mTc-TP3654 was able to detect a number of tumors within 2 hr post injection, including breast and colon cancer. No pharmacologic effects were observed following administration of 5 ug peptide. In humans, approx. 70% of the radioactivity cleared through the kidneys and 20% through the liver (Thakur et al., 2000). For 18F labeling, an analogue of VIP was prepared in which Arg replaced Lys residues at positions 15 and 21. [Arg15, Arg2l]VIP has only one remaining Lys at position 20 as a potential site for amine group modification for radiolabel attachment. [Arg15, Arg21]VIP was coupled to 18F-succinimidyl benzoate. The labeled conjugate bound to breast cancer cells in vitro and was shown to be internalized into the cells. In mice bearing MCF-7 tumors, 18F-PFB-[Arg15, Arg2l]VIP uptake in tumor was 2.46% ID/g at 2 hr. This was higher than all other tissues except kidney, which was 2.61 %ID/g. The levels in other major organs were blood (2.02 ID/g), lung 1.72, liver 0.93, and breast 0.47 %ID/g (Moody et al., 1998). PI666 is an analogue of VIP in which Met17 was replaced with homocysteine linked through its thiol to a short tetrapeptide side chain (Gly-Gly-Cys-Lys) to comprise a chelator for reduced Tc. The unlabeled peptide retained high affinity for VIP receptors on human breast cancer cell membranes, and the oxorhenium complex of the peptide had even better (2x) affinity. At 90 min post injection, uptake in the C-neuOncomouse® murine breast tumor was 0.76 %ID/g. Tumor-to-blood ratio was 3.9 and tumor-to-muscle was 5.3. Blocking reduced uptake to 33% of unblocked value. Uptake in LoVo human colon CA xenograft in mice was 0.55%ID/g, with tumor-to-blood of 2.6 and tumor-to-muscle of 3.7 (Nelson et al., 2000). VIP is potentially very interesting, because receptors for it are found on a wide variety of cancers. It is not a universal tracer, however. Many of the less common neuroendocrine tumors had a lower incidence of VIP expression than SSTR2 expression, including pheochromocytomas, neuroblastomas, and growth hormone producing pituitary adenomas. Medullary thyroid cancer and Ewing's sarcoma did not express VIP receptors (Reubi, 1995a). Despite its promise, major difficulties remain in radiolabeling, pharmacologic activity, and instability in vivo. More stable analogues with antagonist activity are awaited. SUBSTANCE P Substance P (SP) is a regulatory undecapeptide, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met, which belongs to the tachykinin family. Three different tachykinin receptor subtypes (NKl, NK2 and NK3) have been described. Substance P has highest affinity for N K l , which is found in brain, lymphoid tissues, vessels, gut smooth muscle, airway glands and bronchiolar walls (Hennig et al., 1995). Substance P receptors are also found in inflammatory disease and in thymus and salivary glands. In receptor autoradiography of tumor specimens ex vivo, substance P receptors were found to be more abundant than somatostatin receptors on

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

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glioblastoma, medullary thyroid cancer (MTC), non-small cell lung cancer (nSCLC) and carcinoma of pancreas (but incidence is low in the latter two). Also, SP receptors have been found on peritumoral vessels associated with MTC, breast carcinoma, ganglioneuroblastoma, neuroblastoma, nSCLC, colon carcinoma, pancreatic carcinoma and malignant lymphoma (Hennig et al., 1995). Initial radiolabeling was performed by modifying the peptide at its amino terminus, which is not essential for bioactivity (Ozker et al., 2000). [125I-Bolton-Hunter-Arg']-SP and [111in-DTPA-Arg'j-SP bound in vitro and in rats to the following normal tissues known to be SP receptor positive: colon, jejunum, submandibular and parotid glands. The peptides also bound weakly to CA20948 tumors in rats: at 24 hr post injection, specific uptake in tumor was 0.07 %ID/g. The peptide is rapidly metabolized, with an effective Ti/ 2 in blood of 3 minutes. Blood and urine samples contained very little intact peptide (Breeman et al., 1996). In patients who received 2.5 - 5 pg of [111in-DTPA-Arg'j-SP for imaging, all subjects experienced a transient flush. This is indicative of vasodilation, a known pharmacologic effect of substance P. In these clinical studies, [111in-DTPA-Arg'j-SP was degraded rapidly, with a 4-minute half-life of intact labeled peptide in blood. The thymus was visualized in patients who had autoimmune diseases: this is probably indicative of substance P receptor upregulation in immune response. Other sites of inflammation were also visualized (van Hagen et al., 2000). Table 4.

Peptide Ligands for GRP Receptors Sequence

Name GRP (15-28) Neuromedin C Litorin Neuromedin B BOMBESIN [Tyr^bombesin [Lys3]bombesin [DTPA',K3,Y4]bombesin 5Ava-BN(7-14) bombesin(7-13)

Tyr-Pro-Arg-Leu-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2 Gly~Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2 pGlu-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2 Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH2 pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 pGlu-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 pGlu-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 DTPA-Gln-Gln-Lys-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 5Ava-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 pGlu-Trp-Ala-Val-Gly-His-Leu-NH2

GRP receptor antagonists [Tyr5,DPhe6]BN(5-13)NHEt RC-3095 BW2258U89 BIM ¥ = reduced peptide bond (CH2-=NH)

Tyr-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH2CH3 isubutyryl-His-Trp-Ala-Val-DAla-His-Leu-NHCH3 DTpi-Gln-Trp-Ala-Val-Gly-His-LeuW-Leu-NH2 (de-NH2)Phe-Asn-His-Trp-Ala-DAla-Gly-DProW-Phe-NH2 DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NH2

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HANDBOOK OF RADIOPHARMACEUTICALS

BOMBESIN Bombesin (BBN) is an amphibian peptide from the skin of the European fire-bellied toad, Bombina bombina. Bombesin is structurally related to the mammalian peptides Gastrin Releasing Peptide (GRP) and neuromedin B (see Table 4). These are neuropeptides/growth factors with receptors found in CNS, intestines and pancreas. Bombesin, like its related peptides, has a wide variety of pharmacologic actions (Condamine et al., 1998): it acts as a neurotransmitter/neuromodulator by stimulating muscles of the gastrointestinal tract, it stimulates secretion of pancreatic enzymes and gastrointestinal hormones, it enhances the proliferative activity of rat adrenocortical cells, and it acts as a mitogenic agent, displaying growth-factor activity for human small-cell lung cancers and possibly some intestinal cancers. Receptors are overexpressed in human small-cell lung cancer, prostate, breast, gastric, colon and pancreatic cancer, and glioblastoma. Bombesin/GRP agonist analogues may function as paracrine/autocrine growth stimulators in many cancers (Breeman etai, 1999b; Baidoo et al., 1998). For a diagnostic agent, it is obviously desirable to avoid stimulating the growth of cancer, so an antagonist rather than an agonist would be preferred. Bombesin receptor antagonists have been explored as therapeutic agents to arrest tumors which have high densities of BBN receptors. Antagonists to the receptor, such as antibodies, may induce remission in SCLC. Substance P analogues may act as antagonists for the BBN receptor, but the analogues initially tried were of low affinity. Newer highly potent analogues have been developed. It has been found, however, that BBN agonists are internalized following receptor binding, whereas BBN antagonists are not (Breeman et al., 1999b). As a consequence, labeled BBN agonists are able to achieve higher specific uptake in BBN receptor-rich tissues and higher target-to-nontarget ratios. Bombesin contains no cysteines and thus assumes a disordered conformation in solution. Molecular modeling and NMR indicate, however, that in conditions which simulate a receptor surface, bombesin residues Asn6-Met14 assume an a-helical conformation and the N-terminal portion remains linear (Condamine et al., 1998). The C-terminal eight residues are believed to contain the domain responsible for receptor recognition. Small carbonyl modifications in the last two C-terminal residues might be responsible for determining whether a bombesin analogue has agonist or antagonist properties (Condamine et al., 1998). Bombesin(7-13)NH2 was labeled with the N-succinimidyl ester of

I25

l-metaiodobenzoate to place a

metaiodophenyl (mlP) group on the N-terminus of the peptide (Rogers et al., 1997). Radiochemical yield was only about 10%, which was attributed to the more limited availability of primary amines in a small peptide with only an N-terminus compared to multiple lysine sidechains in an antibody which labels up to 60% by this method. However, NHS esters work better with lysine sidechains, and the N-terminal Gin may be readily converted to the cyclic pyroglutamate in aqueous solution (Grant, 1992) so it would be less reactive with the ester.

l25

I-mIP-bombesin(7-13)NH2 was compared with directly iodinated Tyr4-bombesin.

The rate of internalization into BNR-11 cells in vitro was similar, but elution of radioactivity from the cells was fast with directly iodinated peptide and much slower with the more stable mlP conjugate; this is probably related to faster breakdown of the directly iodinated peptide. Uptake in tumors in mice was also improved with the mlP label, as were tumor background ratios (Rogers et al., 1997).

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

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Baidoo et al, conjugated DADT bifunctional chelators to the e-amino group of Lys in [Lys3]bombesin. Radiolabeling was accomplished by ligand exchange from 99mTc-glucoheptonate. Since the active portion of the peptide is the C-terminal 8 amino acids, modification of Lys3 had no effect on functional activity. The peptide could be modified without sidechain protection, since the £-amino group of Lys is the only moiety susceptible to modification under the reaction conditions. Two diaminedithiol (DADT) chelators were used, one (Hx-DADT) providing a neutral Tc(V)oxo core, and the other (Pm-DADT) providing a positively charged chelate core. Syn- and anti-isomers of each of these 99mTc conjugates were separable on HPLC. HPLC purification not only provided single isomers for testing, but also separated radiolabeled material from unlabeled cold peptide, thereby yielding high specific activity material. 99mTc-labeled DADT conjugates had better affinity for the BBN receptor than did unlabeled conjugates or unconjugated peptide (Baidoo et al., 1 998). [Tyr3]bombesin labeled with the neutral Tc core was more lipophilic and in biodistribution studies in normal mice exhibited more liver and intestinal excretion than the same peptide labeled with the positively charged core (Baidoo et al., 1998). However, there was still some hepatobiliary excretion with the positively charged core. In an attempt to reduce lipophilicity further, DTPA was coupled to the amino terminus of [Lys3, Tyr4]bombesin during solid-phase peptide synthesis. The DTPA was not used for bifunctional chelation, but simply to add negative charge to increase hydrophilicity. The chelator Pm-DADT was attached via the £amino group of Lys. Receptor binding in vitro was unaffected by adding the Pm-DADT to the peptide. Biodistribution in mice showed some liver (0.95 %ID/g) and GI tract (2.27 % ID/g) at 60 min, but this was less than without the DTPA (Lin et al., 2000). A tetradentate dithiaphosphine (P2S2) bifunctional chelating agent has also been used for labeling a bombesin analogue (Karra et al., 1999). Bombesin(7-14) was produced with 5-amino valeric acid (5Ava) on the amino terminus, which provided a (CH2)4 spacer to assure placement of the bifunctional chelating agent well away from the peptide 's binding domain. In a preformed chelate approach, the P2S2 BCA was labeled with 99mTc, activated with pentafluorophenol (PFP) and then coupled to the N-terminus of the bombesin analogue with yields >60%. The labeled conjugate was separable from unlabeled peptide by HPLC. In normal mice, uptake was observed in pancreas, which is known to be rich in GRP receptors. This uptake was >90% reversed by blocking with cold bombesin. Excretion of this peptide was 57% renal and 35% hepatic by 4 hr (Karra et al., 1999). In another approach, trisuccin was coupled to the N-terminus of BBN(7-13) during solid phase synthesis. Trisuccsn is planned as a chelator for reduced Re or Tc. Binding in vitro to BNR-1 1 cells transfected with murine GRP receptor showed that [188Re-trisuccin-Gln']BBN(7-13) had slightly lower binding compared to [125I~Tyr4]BBN (Safavy et al., 1997). was placed on N-terminus of BBN(8-14) during solid-phase peptide synthesis, for the purpose of labeling with reduced Tc. However, the resulting complex had excessive hepatobiliary excretion (Okarvi, 2000).

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HANDBOOK OF RADIOPHARMACEUTICALS

Breeman et at. synthesized a number of analogues of bombesin. Of the peptides tested for binding to 7315b rat pituitary tumor cell membranes, [DTPA, Tyr5, DPhe6]BBN(5-13)NHEt, an antagonist, and [DTPA, Pro',Tyr4]BBN, an agonist, had the highest affinities. When labeled with '"in, the agonist, but not the antagonist, was internalized by BBN-receptor positive tumor cells in vitro. Accordingly, the agonist had much higher specific uptake in tumor and receptor-positive normal tissues (pancreas) in rats with 7315b rat pituitary tumor. In addition, the agonist had lower uptake in receptor-negative normal tissues such as liver, lung, and blood (Breeman et al., 1999b). Despite the potential for pharmacologic effects such as gastrointestinal hormone release and smooth muscle contraction, rats injected with up to 100 pg of [Tyr4]bombesin showed no signs of discomfort (Breeman et al., 1999b). Urine from rats injected with ['"in-DTPA, Pro',Tyr4]BBN contained about half the radioactivity in a peptide-bound form, although it was not determined whether this was fully intact peptide or a partially degraded form. Urinary excretion in rats was fairly rapid: 35 % excreted by 1 hour, and 70 % excreted by 24 hr post injection (Breeman et al., 1999a). As with octreotide, the highest specific activity material did not result in the highest uptake in receptor-positive normal tissues; 0.1 pg per rat appeared to give better uptake than higher or lower peptide doses. This is attributed to a possible clustering of ligand-occupied receptors, which may be required for internalization (Breeman et al., 1999a). Uptake of ["'in-DTPA, Pro',Tyr4]BBN in CA20948 pancreatic tumor in rats was 0.17 %ID/g, 90% of which was specific, and uptake in CC531 colon carcinoma in rats was 0.10 %ID/g (about 10% specific). These values are lower than for uptake of '"in-octreotide in SSTR2-positive tumors (Breeman et al., 1999a). a-MELANOCYTE-STIMULATING HORMONE Alpha-melanocyte-stimulating hormone (a-MSH; a-melanotropin; see Table 5) is a peptide hormone which affects pigmentation in peripheral tissues, especially the skin. It is related to and has the same N-terminal tridecapeptide sequence as corticotropin, except that the N-terminus is acetylated in the case of a-MSH. Both a-MSH and corticotropin have a variety of biologic activities in the CNS (Hruby et al., 1993). Receptors for a-MSH are expressed on melanoma cells. Unlike other melanocortins, the human a-MSH receptor is expressed only on melanoma cells and not on normal tissues. Table 5. Peptide Ligands for MSH Receptors Name

Sequence

a-MSH [Nle4,DPhe7]-a-MSH (cyclized through Tc or Re)

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Ac-Cys-Cvs-Glu-His-DPhe-Arg-Trp-Cvs-Lvs-Pro-Val-NH?

NOTE: underlined portion is cyclic

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

663

Certain structural features of ct-MSH are known to be important for receptor binding and activity. N-acetyl and carboxamide terminal groups are important for potency. The primary binding sequence is probably His6Phe7-Arg8-Trp9, while other residues simply modulate the binding activity. DPhe7 substitution for LPhe' increases resistance to proteolytic degradation with no loss of biologic activity. Oxidation of Met4 causes dramatic loss of activity; this can be prevented by substitution of the isosteric Nle4. One of the most potent MSH analogues is [Nle4, DPhe7]-a -MSH, which has an affinity for the a-MSH receptor many fold higher than a-MSH itself (Hruby et al, 1993). A radiolabeled analogue, [i25I-Tyr2,Nle4, DPhe7]a -MSH was shown to be internalized following binding to B16 murine melanoma cells (Adams et al., 1993). a-MSH and [Nle4, DPhe7]-a-MSH were radiolabeled indirectly at Lys11 using N-succinimidyl-3[125I]iodobenzoate (SIB) and directly (at Tyr2) with 131I using lodogen (Garg et al., 1996). In vitro, [Nle4, DPhe7,Lys"-(125I)IBA]-a-MSH (prepared using SIB) had higher binding to murine B-16 melanoma cells (34% vs. 15% in 3 hr) and lower Kd (10 pM vs. 140 pM) than did [Tyr2(131I),Nle4, DPhe7]-a-MSH. This is thought to be due in part to increased stability. In mice, both labels for a-MSH showed rapid tissue clearance, indicative of rapid enzymatic breakdown of the peptide; however, the directly iodinated molecule had higher early retention in normal tissues and higher thyroid and stomach. [Nle4,DPhe7]-a-MSH is less susceptible to proteolytic degradation compared to a-MSH. In mice, [Nle4,DPhe7,Lysu-(125I)IBA]-a-MSH had faster clearance from normal tissues than did [Tyr2(131I),Nle4,DPhe7]-a-MSH. When urine was analyzed, the primary catabolite from [Nle4,DPhe7,Lys"-(i25I)IBA]-a-MSH was the lysine conjugate of I2:TIBA whereas the primary catabolite from [Tyr2(131I),Nle4,DPhe7]-a-MSH was free radioiodide (Garg et al., 1996). Thus, it is clear that stabilizing a peptide against enzymatic degradation is important, but stabilizing a radiolabel, and controlling the biodistribution of radiolabeled catabolites, is also of vital importance. The location of the radiolabel within the peptide is also relevant. Garg et al. suggested that the [i25I-IBALys sl ]a-MSH label gave better binding results because Lys" is closer to the binding domain of the peptide. A label at Tyr2 is cleaved off sooner, and thus such a molecule is less likely to have a label on the portion of the molecule still able to bind to the receptor (Garg et al., 1996). A dimeric analogue of a-MSH was created in which two peptide segments were coupled to a single DTPA for chelating mln. This molecule, DTPA-bis-{[Nle4,Asp5,DPhe7,Lysi0]a-MSH (4-10)}, was labeled with 111 In and compared with a monomeric analogue, lllIn-DTPA-[Nle4,Asp5,DPhe7,Lys(bis-DHP)10]a -MSH (410). In mice bearing B16-F1 melanoma tumors, the dimeric molecule had higher tumor binding (total binding 1.26 %ID/g at 4 hr, vs. 0.64 %ID/g for the monomeric analogue); however, the monomeric analogue had lower normal tissue background (Bagutti, 1993). The dimeric analogue was tested in patients with malignant melanoma, and succeeded in imaging 41 of 46 known lesions within 3-4 hr after injection. It was noted, however, that substantial liver uptake precluded direct imaging of liver metastases without background subtraction techniques (Bard et al., 1993). The tissue clearance of a-MSH analogues may be rapid enough to consider using the short-lived label i8F. [Nle4, DPhe7]-a-MSH was also labeled with a radiofluorinated analog of SIB, [I8F]SFB, for use with PET (Vaidyanathan & Zalutsky, 1997). Radiochemical yields were routinely about 80% (decay-corrected). In

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HANDBOOK OF RADIOPHARMACEUTICALS

normal mice, [Nle4, DPhe7,Lys"-(18F)FBA]-a-MSH had similar biodistribution to its iodinated analogue, except that liver and intestinal activity were lower for the I8F analogue. There was little indication of defluorination. Natural a-MSH is believed to have a beta-turn in the molecule, such that Phe7 is at the apex of the turn. Potent analogues have been created by constraining this turn using disulfide or lactam rings (Hruby et al., 1993). In a novel approach to radiolabeling peptides, Giblin et al. designed an a-MSH analogue which is cyclized through site-specific Re or Tc metal coordination (Giblin et al.t 1998). In the most potent of these analogues, the peptide Ac-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Lys-Pro-Val-NH2 was labeled with Re by exchange from Re-glucoheptonate in 62% methanol, pH 8-9 at 65-70°C for 1 hr. The peptide was purified by HPLC. Both Re(V) and Tc(V) should assume square planar coordination geometry. The two N-terminal Cys thiolates, in combination with the intervening amide nitrogen, form three comers of the square plane, while the Cys10 thiolate is the remaining donor atom. This results in a tricyclic molecule, with rings of 6, 5, and 24 members. When prepared with stable rhenium, the complex showed no degradation over 3 weeks, and had a Kd for a-MSH receptors of 2.9 nM. Biodistribution of the "Tc-labeled analogues in mice bearing melanomas showed tumor uptakes of 10.9 %ID/g at 1 hr and 9.5 %ID/g at 4 hr. Tumor blood ratios were 6.8 at 1 hr and 15.3 at 4 hr post injection. Excretion was rapid and predominantly renal, with >60% of the administered dose present in the urine at 30 min. Some kidney retention (5% at 1 hr) was seen (Giblin et al., 1998). This approach provided exciting results but the labeling may be tricky to perform consistently and requires purification before use. There are differences between murine and human melanoma cells with respect to binding a-MSH. With murine cells, Kds are in the range 0.3-1.0 nM with 5,000 to 20,000 sites/cell. Human melanoma cells have about 1/10 the receptor density and up to 5-fold higher affinity. On murine melanoma cells, the a-MSH receptors internalize bound ligand rapidly; however, human melanoma cells did not internalize bound '25I-aMSH (Siegrist et al., 1989). This suggests that the results of imaging studies performed with a-MSH in mice with murine melanomas should be interpreted with caution in predicting efficacy in humans with melanoma. CALCITONIN Calcitonin is a 32-amino acid neuropeptide (see Table 6) that regulates calcium levels. Receptors for calcitonin are found in normal osteoclasts, lymphocytes, kidney, brain, testes and prostate. Receptors are found in increased density in osteolytic bone disease (e.g., metastatic bone cancers and Paget's disease), breast cancers, bronchial cancer, prostate cancer, malignant lymphoid tissue, and medullary carcinoma of the thyroid (Blower et al., 1998). In one study, 7 of 8 breast carcinoma cell lines tested expressed calcitonin receptors, while a normal breast tissue cell line (HS578T) did not (Nelson et al., 2000).

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

665

Table 6. Peptide Ligands for the Calcitonin Receptor Name | Salmon Calcitonin

Sequence Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-GlnGlu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2

Human Calcitonin Cys-Gly-Asn-Leu-Ser-Thr-Cvs-Met-Leu-Glv-Thr-Tyr-Thr-GlnAsp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 PI 410

CH2CO-Ser-Asn-Leu-Ser-Thr-Hcy-Val-Leu-Glv-Lys-Leu-SerC(CH2CO(eLys)-Gly-Cys-Glu-amide)Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2

NOTE; underlined portions are cyclic Salmon calcitonin, which has higher affinity for the human calcitonin receptor than human calcitonin, is used as a therapeutic drug for inhibiting bone resorption. Compared with human calcitonin, salmon calcitonin is also less likely to form the thick fibrillar aggregates which are characteristic of human calcitonin and make it difficult to handle (Moriarty et al., 1998). Salmon calcitonin still presents some difficulties in handling, including sticking to container walls, column packing and sterilizing filters (Blower et al., 1998). Salmon calcitonin contains a single disulfide crosslink, joining residues 1-7 in a well-defined loop which is believed to interact with the portion of the receptor which is responsible for activation of adenylate cyclase. Residues 8-22 assume a helical conformation and are responsible for most of calcitonin's interaction with the transmembrane loop region of the receptor (Stroop et al., 1996). The Cys'-Cys7 ring is in close association with the helix of residues 8-22, and the C-terminal decapeptide folds back toward the core, forming a loose loop (Meyer etal,, 1991). Blower et al. labeled salmon calcitonin at Tyr22 using Chloramine-T. Use of metabisulfite to stop the reaction had to be avoided because it caused reduction of the disulfide bond of the N-terminal loop. Triton X-100 was helpful in minimizing losses due to peptide adsorption to surfaces, but sterilization by membrane filtration was still not possible without significant loss of peptide. The product of the labeling, di-iodinated calcitonin, retained in vitro biologic activity compared to unlabeled peptide. In normal mice, the main organ accumulating tiacer was kidney, and to a lesser extent liver and lungs. In patients with cancer, some known sites of Paget's disease accumulated tracer. The radiopharmaceutical had rapid blood clearance, with an initial T 1/2 of 3-7 min. Excretion was primarily by the kidneys, but liver and lungs were seen in whole-body images. Extensive muscle (soft tissue) background was seen. This is consistent with very rapid deiodination of the labeled peptide, with significant amounts of free iodide and iodotyrosine even as early as 15 minutes pos< injection (Blower et al., 1998). A

99m

Tc-labeled calcitonin analogue, is under development.

This peptide, PI410, differs from salmon

calcitonin in the following ways: The N-terminal loop structure, instead of being cyclized by a disulfide

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HANDBOOK OF RADIOPHARMACEUTICALS

bond between Cys1 and Cys7, is cyclized with a sulfide between CH2- and die thiol of homocysteine. A chelating site for reduced Tc was created by a Gly-Cys-Glu sequence in a branch off the main peptide chain. [""^TcOJPl 410 had Kd =2.5 nM for T47D human breast cancer cells. In mice bearing MCF-7 human breast xenografts, tumor uptake was 0.75 %ID/g at 90 min, compared with normal tissue levels of lung 0.35, liver 0.98, kidney 5.3 and GI tract 2.89 %ID/g. Average Tumor/Muscle and Tumor/Blood were 4.5 and 5.7 (Nelson et al., 2000). NEUROTENSINS Neurotensin (NT; see Table 7) is a linear peptide that is found in high concentrations in ileum and hypothalamus, and induces various physiologic effects such as hypotension, analgesia, gut contraction, and increase of vascular permeability (Sefler et al., 1995). Receptors for neurotensin are expressed on pancreatic and prostate cancer (Hua et al., 1996). Neurotensins are internalized after receptor binding (Schubiger et al., 1999). Table 7. Peptide Ligands for Neurotensin Receptors Name

Sequence

Neurotensin NT-I NT-II NT-Ill NT-IV NT-V NT-VI FB-P3

pGlu-Leu-Tyr-Gln-Asn-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-fle-Leu His-Arg-Arg-Pro-Tyr-Ile-Leu (Na-His)-Ac-Arg-Arg-Pro-Tyr-Ile-Leu His-(N-Me)Arg-Lys-Pro-Tyr-Ile-Leu His-Lys¥-Arg-Pro-Tyr-Ile-Leu (Na-His)-Ac-ArgW-Arg-Pro-Tyr-Ile-Leu (Na-His)-Ac-LysW-Arg-Pro-Tyr-Ile-Leu 4-fluorobenzoyl-ArgW-Arg-Pro-Phe-Tle-Leu

W = reduced peptide bond (CH2-NH) The C-terminal hexapeptide, Arg-Arg-Pro-Tyr-Ile-Leu or its closely related analogue, Arg-Lys-Pro-Tyr-IleLeu are the shortest sequences derived from the native peptide which retain binding affinity for the receptor (Sefler et al., 1995). In the native peptide, positions Arg8-Arg9 and Pro10-Tyr'' are particularly susceptible to cleavage by endoproteinases. Schubiger et al. substituted N-methyl arginine for Arg or reduced peptide bonds (CH2=NH) in selected locations to minimize enzymatic degradation (Schubiger et al., 1999). The analogues also included just the 6 critical residues, plus a His or (Na-His-)Ac at the N-terminal position for radiolabeling with Tc-tricarbonyl. For radiolabeling by the Tc-tricarbonyl method, an N-terminal His residue provides bidentate (N,N) chelation, whereas (Na-His-)Ac at the amino terminal position provides tridentate (N,N,O) chelation (Schubiger et al., 1999).

Peptides with N-terminal His (bidentate chelation) exhibited plasma protein

binding, whereas peptides with N-terminal (Na-His-)Ac had no protein binding.

RADIOLABELED PEPTIDES FOR TUMOR IMAGING

667

Generally, the truncated and stabilized analogues retained the biologic activity of the parent tridecapeptide. The single exception was NT-VI which exhibited a 5- fold decrease in affinity: apparently it could not tolerate both the Lys8 and the (Na-His-)Ac7 substitutions (Schubiger et al., 1999). Redistribution studies in normal mice at 24 hr post injection suggested that stabilization improved biodistribution: more of the radioactivity was excreted by the kidneys. Stabilized peptides had slightly more liver uptake; however, some of this may have been due to plasma protein binding, as use of tridentate chelation reduced this liver uptake. Fluorine-18 labeled analogues of NT(8-13) have also been explored (Bergmann et al., 2000). The most promising of these was a double-stabilized Arg-^-Arg-Pro-Phe-Tle-Leu labeled at the N-terminus with 4fluorobenzoate. This peptide, FB-P3, was tested in nude mice bearing HT-29 or WiBr tumors, where it exhibited tumor uptake of 3.5% ID/g at 10 min post injection. Excretory organs accumulated considerable radiotracer, preventing imaging of lesions which were close to these organs, so further structural optimization is required.

CCK AND GASTRIN CCK (cholesytokinin) and gastrin (Table 8) act as neurotransmitters in the CNS, as regulators of various functions in the gastrointestinal tract, and as stimulatory growth factors in several cancers, such as colon cancer and gastric cancers. They are structurally related in that they share the same C-terminal five amino acids, which is the active site for binding to their common receptor, termed CCK-B. CCK binds to two types of CCK receptors, CCK-A and CCK-B, whereas gastrin binds avidly only to CCK-B. In normal tissues, the CCK-B receptor is found in gut mucosa and brain, whereas CCK-A receptors are found in gallbladder, pancreas and brain (Reubi et al., 1997). Medullary thyroid carcinoma (MTC), although it is a neuroendocrine tumor, does not have consistent expression of somatostatin receptors of a subtype which binds octreotide analogs. It has been reported, however, that MTC expresses CCK-B/gastrin receptors (Reubi et al., 1997). In autoradiographic studies, it has been found that >90% of MTC specimens expressed CCK-B/gastrin receptors, as did a high percentage of other tumor types, including SCLC, astrocytomas and stromal ovarian cancers (Reubi et al., 1997).

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HANDBOOK OF RADIOPHARMACEUTICALS

Table 8. Peptide Ligands for CCK-B Receptor Name

Sequence

Gastrin-related human gastrin-I Biggastrin minigastrin

pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 pGlu-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser-LysLys-Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2

[DGlu'lminigastrin [DLeu ^minigastrin

DGlu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 DLeu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2

CCK-related CCK-8

Asp-Tyr[S03H]-Met-Gly-Trp-Met-Asp-Phe-NH2

CCK-8 (not sulfated)

Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 DTyr-Gly-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2

Gastrin-I is a 17-amino acid peptide which is specific for the CCK-B/gastrin receptor, with an affinity constant in the subnanomolar range; its affinity for CCK-A receptor is more than four orders of magnitude lower (de Jong et al., 1999). Behr et al. labeled human gastrin-I with 13I I using the lodogen method [ I 3 I ITyr12]gastrin-I was tested in nude mice bearing medullary thyroid carcinoma (Behr et al., 1998). At 1 hr, major uptake was seen in tumor, gallbladder, kidneys, stomach and pancreas; uptake in tumor, stomach and pancreas could be blocked by high doses of unlabeled peptide. Tumor uptake was 8.9 %ID/g at 1 hr. Initial studies in a patient showed uptake of labeled peptide at possible metastatic sites in mediastinum and lung (Behr era/., 1998). In subsequent studies, Behr et al. evaluated a number of gastrin derivatives of various lengths, labeled with radioiodine (Behr et al., 1999). Of these, the analogues with the best affinity and selectivity for the CCK-B receptor, with relatively low renal retention, were minigastrin (a 13-amino acid peptide) and the D-Leu1 analogue of minigastrin (see Table 8). These were modified at the N-terminus with DTPA and labeled with '"in. In nude mice bearing human MTC tumors, '"in-DTPA-minigastrin had tumor uptake of 5 %ID/g at 1 hr. Uptake was also seen in stomach, which is rich in CCK-B receptors. Blood clearance was rapid, with primarily renal excretion and maximal kidney uptake of 45% ID/g. Some hepatobiliary excretion was observed. In initial studies in three patients with metastatic MTC, "'in-DTPA-minigastrin targeted receptors in the stomach and known metastatic lesions.

Two patients reported transient symptoms of nausea or

abdominal discomfort after the administration of the dose (0.5 mg/kg) (Behr et al., 1999).

In a trial of 35

patients with metastatic MTC, all known tumor sites were visualized, as early as 1 hr post injection. Occult lesions were detected in 14 subjects (Behr et al., 2000).

RADIGLABELED PEPTJDES FOR TUMOR IMAGING

669

CCK is 33-amino acid peptide, with a sulfated Tyr in position 27 which is essential for retention of high affinity for both CCK-A and CCK-B receptors. This is true also of the octapeptide fragment CCK(26-33), referred to as CCK-8, which consists of the C-terminus of the molecule and includes the active binding site.

125

I-DTyr-Gly-Asp-Tyr(SO3H)-Nle<jly-

Trp~Me~Asp-Phe-NH2 recognizes both CCK-A and CCK-B receptors, whereas the nonsulfated analogue 125I-DTyr-GIyAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NHa specifically recognizes CCK-B (Reubi et al. 1997). More recently, certain unsulfated CCK analogues have been found which bind selectively to CCK-B receptors (Reubi et al., 1998). For imaging and therapy, a radiometal label is preferred, so DTyr-Gly-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-PheNH2 and other nonsulfated analogues were conjugated with DTP A. All peptides retained activity, provided the chelator was attached at the N-terminus rather than in other positions in the peptide.

Substitution of

DOTA for DTPA had no effect on the activity. ''' In-DTPA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 was stable in plasma and bound well to tumor specimens expressing CCK, including MTCs, SCLCs, astrocytomas, gastroenteropancreatic tumors, and stromal ovarian tumors. In normal rats, blood clearance was very rapid, and excretion was primarily through the kidneys. Aside from kidneys and bladder, the main organ seen in images was the stomach, which is a site of CCK-B receptor binding. HPLC analysis of urine at 1 hi post injection showed that there was no intact peptide present (Reubi et al., 1998). lu

In-DTPA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2

was internalized into CCK-B

receptor-positive

AR42J rat pancreatic tumor cells in vitro. In rats bearing CA20848 tumors, tumor uptake of the "!!n-DOTA analogue averaged 0.16 %ID/g at 1 hr post injection, decreasing slightly to 0.082 %ID/g at 24 hr. Tumor uptake of the !11In-DTPA peptide was similar, 0.094 %ID/g at 24 hr. Levels in normal tissues were lower, except for kidney (0.322 %ID/g at 24 hr); however, kidney levels were lower than are typical with

Ui

ln-

octreotide, possibly because the peptide has net negative charge which may have a lower uptake by the negatively charged membranes of renal tubular cells. Tumor: blood averaged 16.4 and 18.8 at 24 hr, for the 11

'in-DOTA and 1HIn-DTPA analogues, respectively. An initial clinical image demonstrating clear uptake of

H1

In-DTPA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 in MTC at 48 hours emphasized that the radiotracer is retained at the tumor site, owing to the intemalization of the radioligand and the residualization of the radiolabel (de Jong et al,, 1999). MARKERS OF TUMOR ANGIOGENESIS The approaches above use a direct strategy for detecting tumors: binding to receptors directly on the cancer cells. An inherent limitation of this approach is the instability of some cancer cells with respect to receptor expression. An alternative is the indirect strategy of targeting the evolving vasculature that nourishes the growing tumor (Keshet & Ben-Sasson, 1999). Because all solid tumors are angiogenesis-dependent, this approach to diagnosis and therapy of tumors should be applicable to tumors of many cell types - a more universal tumor-imaging approach. Folkman was the first to propose that the expansion of a tumor beyond the size of a ^ew cubic millimeters depends on the formation of blood vessels to provide the growing tumor with oxygen and essential nutrients (Folkman, 1971). It is now believed that the endothelial cells in tumor vasculature also support tumor survival and proliferation by providing growth factors and other cytokines. A distinguishing characteristic of

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HANDBOOK OF RADIOPHARMACEUTICALS

tumor blood vessels is that they are relatively immature, and actively growing. Mature blood vessels are generally quiescent, with only 0.01% of endothelial cells undergoing cell division at a given time. In tumors, however, the fraction of cycling cells might be 2-3 orders of magnitude higher (Keshet & Ben-Sasson, 1999). INTEGRINS The integrin family of cell-adhesion receptors has been reviewed by numerous authors (Cox et al., 1994; Hynes, 1992; Ruoslahti, 1991).

Integrins are heterodimers composed of one alpha and one beta subunit.

There are about 8 beta subunits which form various combinations with about 16 alpha subunits. Both subunits are glycoproteins which are anchored in the cell membrane. The amino terminus and the bulk of the peptide chain are extracellular, and a short segment of the C-terminus is intracellular. The extracellular portions of the alpha and beta chains interact noncovalently. When examined by electron microscopy, the extracellular portion of the integrin appears as a globular head composed of both subunits, connected to the cell membrane by two stalks. Ligand binding is dependent upon divalent cations, and may involve regions of both subunits. A characteristic of integrins is their ability to convert extracellular ligand binding into activation of intracellular processes (outside-in signaling) as well as employing intracellular processes to convert an integrin from an inactive state to an active state (inside-out signaling). Different ligands which bind to a common integrin may trigger different intracellular responses (Hynes, 1992). Some of the integrins are closely related: individual integrins can often bind more than one native ligand, and a given ligand often can bind to more than one integrin. One cell adhesion molecule, fibronectin, contains an RGD (Arg-Gly-Asp) sequence which is responsible for its binding to its receptor, o3{M (Pierschbacher & Ruoslahti, 1984). Since then, it has been discovered that a large number of macromolecular ligands for integrins contain RGD in their binding domains. It should be noted that despite this common binding motif, macromolecules containing RGD bind to different integrins with different affinities. Similarly, multiple ligands all containing RGD have different affinities for a given integrin. Some data suggest that the nature of the residues immediately flanking the RGD are critical for defining the conformation of the binding domain. The Ovp3 integrin is found on many types of cells. It is found predominantly on endothelial cells, but is also found on platelets, vascular smooth muscle cells, monocytes, macrophages, osteoclasts, and some B-cells (Shattil, 1995). It binds ligands containing an RGD motif, as does its closely related integrin, o^|b33, which is found exclusively on platelets and megakaryocytes. Both of these integrins bind the ligands fibrinogen, fibronectin, vonWillebrand factor, vitronectin and thrombospondin. In addition, 0^5 can bind osteopontin, denatured collagen, the penton base of adenovirus, laminin, tenascin, thrombin, and bone sialoprotein (Brassard et al,. 1999; Shattil, 1995). 0^5 has been shown to be involved in cell adhesion, tumor metastasis, and bone resorption. This receptor is found on certain cancer cells, including melanoma, glioblastoma and osteosarcoma (Albelda et al., 1990; Cox et al., 1994; Gladson & Cheresh, 1991). It is found on macrophages and is involved in phagocytosis of cells undergoing apoptosis (Cox et al., 1994).

RADIOLABELED PEPTJDES FOR TUMOR IMAGING

671

Expression of 0^3 on the surface of endothelial cells increases during angiogenesis or during an inflammatory response (Felding-Habermann & Cheresh, 1993). Angiogenesis is the term for sprouting of new blood vessels from existing ones. This process requires endothelial cell and smooth muscle cell invasion, migration and proliferation. In addition to being essential for wound repair, angiogenesis is critical for the growth of tumors. The expression of ctv integrins is normally low in microvasculature but increases on vascular sprouts. It has been shown that angiogenesis can be prevented by anti-ctvj33 antibodies or by cyclic RGD-containing peptides. Interestingly, blockade 0^3 of receptors caused apoptosis of actively proliferating vascular cells but not pre-existing vascular cells (Brooks et al., 1994). It should be noted that a variety of integrins have been found on the surface of tumor cells. For example, melanoma cells express avp3, otiPi, a$\ and ctsPi. Expression of fa integrins was restricted exclusively to melanoma cells in the vertical growth phase and metastatic lesions, the most agressive phases of the malignant process (Albelda et al., 1990). 0^5 is found on lung carcinoma cells, and Oypg is found on some cancer cells (Cox et al., 1994). The focus of this chapter will be restricted to o^Pa, because it is critical to tumor angiogenesis. PEPTIDE LIGANDS FOR INTEGRIN ctvp3 There are many native ligands for ctvPs, and all cross-react with other integrins, for example ctnbPs- The native ligands contain an RGD motif at a bend in a loop structure or a flexible coiled conformation in the polypeptide chain. There has been a great deal of interest in creating synthetic peptides which function as antagonists for integrins, and the focus of this research has been to synthesize small peptides containing RGD (see Table 9). It has been found that constraining the mobility of the RGD in a cyclized pentapeptide or hexapeptide has resulted in the highest potencies in vitro. Interestingly, in numerous studies seeking to develop ligands for the ttnbPs integrin, it was found that small peptides could be more selective for asibp3 over avp3 (or vice-versa) than the native ligands. The large native ligands apparently are flexible in the region of the RGD and are able to adjust to various receptor binding pockets, whereas the constrained cyclic structures are less able to accommodate to different receptor dimensions. The data suggest that the binding pocket in the avPs receptor is narrower and less flexible than the binding pocket in (XnbPs.

Therefore, cyclic

pentapeptides are preferred over cyclic hexapeptides, and the most selective of one tested series, cyclo(RGDfV), had the sharpest gamma turn at Gly2, resulting in the smallest spacing between the beta carbons of Arg1 and Asp3 (Pfaff et al., 1994). Other investigators confirmed the Oyp3 specificity of narrow ligands, with cyclo(RGDRGD), which also has very small Arg-Asp spacing (Burgess, 1996). In phage display studies using a library of RGD-containing peptides to find cyclic peptides specific for 0^3, two lead peptides were found: cyclo(CNGRC) and the double-cyclic ACDCRGDCFC ("RGD-4C") (Koivunen et al., 1995). Both of these peptides have been shown to target KS1767 cells and were internalized following binding (Ellerby et al., 1999). It is not known if o^,p3 bound peptides are internalized after binding to endothelial cells. Cyclo(RGDiV) recognizes both Ovp3 and o\,b5 (Pfaff et al., 1994) and abolished angiogenesis in a chick

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HANDBOOK OF RADIOPHARMACEUTICALS

chorioallantoic membrane (CAM) model of angiogenesis (Friedlander, 1995). It also disrupted tumorinduced angiogenesis and promoted regression of human tumors grown in a CAM model (Brooks et al., 1994). Haubner et al. (Haubner et al., 1999) synthesized tyrosine-containing analogues of c(RGDfV), with the tyrosine in either position 4 or 5: c(RGDyV) and c(RGDfY). Each of these peptides is cyclized by an amide bond.

Both modified peptides and their iodinated analogues retained nanomolar affinity for the 0^3

receptor.

The peptides were directly labeled with radioiodine for initial evaluation. Both labeled and

unlabeled peptides were highly selective for OypV with potency for inhibiting vitronectin binding to OvPa about 4000 times as high as for inhibiting fibrinogen binding to a.nbp3.

In mice bearing subcutaneous

melanoma (human M21) or osteosarcoma, maximal tumor uptake of 2.07%ID/g and 3.5 %ID/g occurred at 10 min post injection of the [I25I-DTyr4]c(RGDyV); tumor levels declined thereafter. The peptide had rapid blood disappearance. The primary route of excretion was hepatobiliary, with liver levels at 19-22% ID/g by 10 min post injection. The uptake in tumor was mostly blockable by excess peptide and therefore receptorspecific. Immunohistochemistry of sections of tumor from the mice indicated positive, specific staining for cXv33. Studies with a mammary carcinoma implanted in mice showed lower tumor uptake (1.8 %ID/g at 10 min) which was not blockable and therefore probably not receptor-mediated (Haubner et al., 1999). The second peptide analogue, [125I-Tyr5]c(RGDfY), was even more lipophilic, with lower tumor uptakes in the same models, so it was considered unsuitable. Table 9. Peptide Ligands for ctvPs Receptors Name

Sequence

c(RGDfV) c(RGDfY) c(RGDfK) c(RGDyV) c(RGDyK) c(RGDRGD) RGD-4C c(CRGDGWC) c(CRRETAWAC) c(CNGRC)

Arg-Glv-Asp-DPhe-Val Arg-Glv-Asp-DPhe-Tvr Arg-Glv-Asp-DPhe-Lvs Arg-Glv-Asp-DTvr-Val Arg-Glv-Asp-DTvr-Lvs Arg-Glv-Asp-Arg-Gly-Asp Cvs-Asp-Cvs-Arg-Glv-Asp-Cvs-Phe-Cvs Cvs-Arg-Glv-Asp-Glv-Trp-Cvs Cvs-Arg-Arg-Glu-Thr-Arg-Trp-Arg-Cvs Cvs-Asn-Glv-Arg-Cvs

NOTE: underlined portions are cyclic It should be noted that human osteosarcoma contains Ovf33 receptors directly on the tumor cells as well as on the blood vessels (Pasqualini et al., 1996). Thus, the binding observed with osteosarcoma may be primarily on tumor cells. Also, direct binding to tumor cells may give advantage to small peptides because bound ligand may be internalized. In contrast, it is not known if bound peptide is internalized into endothelial cells

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673

after binding; if it is not, the bound ligand may be susceptible to reversal of binding or displacement by endogenous ligands in the blood. In the series of peptides based on c(RGDfY), it was found that the nature of the amino acid in position 5 was not critical for activity. Therefore, this position was selected for placement of lysine (K) to provide a primary amine functionality for coupling other groups. Initially, DTPA was coupled to c(RGDyK) (de Jong et al, 2000b). This peptide can be labeled with either radioiodine or radioindium, and is expected to be more hydrophilic than c(RGDyV). In vitro autoradiographic and immunohistochemistry assays with human tumor specimens showed specific binding of [125I-DTyr4,DTPA-Lys5]c([RGDyK) to Ovp3 receptors in a majority of human cancer specimens, including prostate, breast, bladder, lung, colorectal, esophageal and adrenal cell carcinomas and neuroblastoma. In addition, 2 of 3 carcinoids were positive, but retinoblastoma and choroid melanoma were negative (Van Hagen et al., 2000). [125I-DTyr4,DTPA-Lys5]c([RGDyK> also bound to and internalized in human carcinoid Bon cells and rat pancreatic CA20948 tumor cells. When [mIn-DTPA-Lys5]c(RGDyK) was administered IV to rats bearing rat pancreatic CA20948 tumors, receptorspecific tumor uptake was observed. Excretion was through the kidneys, in contrast to [!25IDTyr4]e(RGDyK) (the same peptide without DTPA), which was cleared mainly by the liver (de Jong et al., 2000b). Other modifications of the lysine sidechain in c(RGDfK) were performed to provide chelating agents for Tc, Re and Y. [DKCK-N -Lys5]c(RGDfK) was labeled with "mTc or 188Re, and [DOTA-N£-Lys5]-c(RGDfK) was labeled with ^Y. In osteosarcoma-bearing mice, fast blood clearance and predominantly renal excretion were seen for all three peptide conjugates. At 4 hr, tumor uptake was 1.5, 2.5 and 1 % ID/g for 99mTc, 188Re and 90Y labeled peptides, respectively. Tumor: blood ratios were 5.3, 3.9, and 48, and tumor muscle ratios were 6.0, 13.2, and 10, respectively (Bock et al., 2000). For PET, an I8F~labeled version of c(RGDfK) was developed. In order to minimize lipophilicity, the e-amino group of Lys5 was modified with a sugar amino acid (SAA). An amine-protected SAA was coupled to the lysine sidechain of the peptide. [SAA-Lys5]c(RGDfK) was labeled by coupling with 2-[18F]Fluoropropionic acid 4-nitrophenylester to the amine group of the SAA moiety. In osteosarcoma-bearing mice, tumor uptake of the l8F-glycated peptide was 2.9% ID/g at 10 min and 1.7 %ID/g at 2 hr. Liver and kidney at 2 hr contained 2.0 and 2.2 %ID/g. Uptake in adrenals and intestines were also higher than tumor, but other organs were lower. Tumor/blood and tumor/muscle ratios at 2 hr were 13 and 6 (Haubner et al,, 2000). RP593 is a dimer containing two c(RGDfK) binding loops and a single HYNIC moiety for labeling with 99m Tc. The Tc labeling method used tricine and TPPTS as coligands (Rajopadhye et al., 2000). In nude mice bearing OVCAR-3 human ovarian carcinomas, tumor uptake was 5.5% ID/g at 1 hr, and tumor: blood at 2 hr was 9:1 (Janssen et al., 2000). This peptide is an antagonist for both 0^3 and 0^5, with low reactivity with OvPi or oiiibPs (Barrett et al., 2000). In spontaneous tumor model Neu-Oncomouse®, tumor uptakes were 3.4 and 1.5% ID/g at 2 and 24 hr. In this model, uptake was lower for sestamibi (1.1% ID/g at 2 hr) and scrambled peptide (0.4 %ID/g at 2 hr). RP593 was excreted predominantly by the kidneys, with some renal retention (14% and 6% ID/g at 2 and 24 hr). In subsequent studies, the dimer containing two c(RGDfK) binding loops was compared with a monomer containing only one c(RGDfK). Both peptides were labeled with "rnTc using a HYNIC moiety. There was very little difference in tumor uptake in OVCAR-3 tumors between the monomer and dimer (Janssen et al., 2001), suggesting that the high uptake may be related to the

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model rather than to an enhancement caused by having two binding moieties. In the studies above, it is not clear where the peptides are binding in tumor models. Specifically, are the radioligands binding to receptors on endothelial cells or to 0^3 receptors on the surface of tumor cells? More work needs to be done in this area. SUMMARY The area of labeled peptides for cancer imaging has generated tremendous excitement in the last ten years, as judged by the number of investigations reviewed in this chapter. The area is still fairly immature. Although two radiopharmaceuticals ('"in-DTPA-octreotide and """Tc-depreotide) are currently approved for routine clinical use, they are applicable only to a select group of cancers. There are many challenges ahead for developing improved peptide radiopharmaceuticals for optimal imaging of other cancers. Besides fundamental issues of peptide receptor recognition, peptide stability in vivo and the potential for pharmacologic side-effects, there is a major challenge for choosing the most appropriate radiolabeling technique. It is clear that for a single peptide, variations in labeling approach can result in dramatic differences in tumor targeting and background clearance. ACKNOWLEDGEMENTS The author would like to thank the National Heart, Lung and Blood Institute (HL 54578) for financial support.

GLOSSARY ct-MSH Boc CPTA CpTT DCCI DFO DTPA DOTA DKCK EDTA HABt HOBt HPLC HYNIC IBA MAb MAG3

Alpha-melanocyte-stimulating hormone rm-butyloxycarbonyl 4-[( 1,4,8,11 -tetraazacyclotetradec-1 -yl) methyl] benzoic acid cyclopentadienyltricarbonyltechnetium dicyclohexylcarbodiimide desferrioxamine diethylene triamine pentaacetic acid 1,4,8,11 -tetraazacyclododecane-N,N ',N",N" tetraacetic acid Asp-Lys-Cys-Lys ethylenediaminetetraacetic acid hydroxyazabenzotriazole hydroxybenzotriazole High Performance Liquid Chromatography 6-hydrazinopyridine-3-carboxylic acid iodobenzoic acid monoclonal antibody mercaptoacetyl-Gly-Gly-Gly

RADIOLABELED PEPTIDES FOR TUMOR IMAGING MIB OC PACAP PET PnAO SIB SPECT TATE Tc-tricaibonyl TETA TOG VIP Y3-TATE

meta-iodobenzoate octreotide pituitary adenylate cyclase activating polypeptide Positron Emission Tomography propyleneamine oxime succinimidyl iodobenzoate Single Photon Emission Computed Tomography octreotate /ac[99mTc(CO)3(H20)3]+ 1,4,8,11 -tetraazacyclotetradecane-N,N',N",N'" tetraacetic acid [Tyr3]octreotide vasoactive intestinal peptide [TyrVctreotate

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Moriarty DF, Vagts S and Raleigh DP (1998) A role for the Oterminus of salmon calcitonin in aggregation and gel formation: A comparitive study of C-terminal fragments of human and salmon calcitonin. Biochem. Biophys. Res. Commun., 245, 344–348. Nelson CA, Moyer BR, Pearson DA, Manchanda R, Wilson DM, Rutkowski JV, Lister-James J and Dean RT (2000) Radiolabeled P1 470, a calcitonin analog, targets human breast cancer xenografts in nude mice. Nucl. Med. Commun., 21, 575. Oberg K (1996) Neuroendocrine gastrointestinal tumours. Ann. Oncol., 7, 453–463. Okarvi SM (2000) Synthesis and biological evaluation of bombesin peptide analogs radiolabelled with Tc99m. Nucl. Med. Commun., 21, 576. Ozker KS, Krasnow AZ and Hellman RS (2000) 99mTe-labeled substance-P (SP) analogues for SP receptor imaging.J Nucl. Med., 41,246P. Pallela VR, Thakur ML, Chakder S and Rattan S (1999) 99mTc-labeled vasoactive intestinal peptide receptor agonist: functional studies. J. Nucl. Med., 40, 352–60. Pasqualini R, Bourdoulous S, Koivunen E, Woods VL and Ruoslahti E (1996) A polymeric form of fibronectin has antimetastatic effects against multiple tumor types. Nature Medicine, 2, 1197–1203. Pearson DA, Lister-James J, McBride WJ, Wilson D, Martel LJ, Civitello ER, Taylor JE, Moyer BR and Dean RT (1996) Somatostatin receptor-binding peptides labeled with technetium-99m: Chemistry and initial biological studies. J. Med. Chem., 39, 1361–1371. Perryman AL, Zhen C, Jurisson SS and Quinn TP (1999) Design and synthesis of technetium and rheniumcyclized somatostatin analogs. J. Labelled Cpd. Radiopharm., 42 Suppl 1, S156–S157. Pfaff M, Tangemann K, Muller B, Gurrath M, Muller G, Kessler H, Timpl R and Engel J (1994) Selective recognition of cyclic RGD peptides of NMR defined conformation by allbp3, 0^3 and OsPi integrins. J. Biol. Chem., 269, 20233–20238. Pierschbacher MD and Ruoslahti E (1984) Cell attachment activity of fibronectin can be duplicated by small i synthetic fragments of the molecule. Nature, 309, 30-33. Presky DH and Schonbrunn A (1988) Somatostatin pretreatment increases the number of somatostatin receptors on GH4C] pitutary cells and does not reduce cellular responsiveness to somatostatin. J. BioLChem.,263,714–72l. Raderer M, Becherer A, Kurtaran A, Angelberger P, Li S, Leimer M, Weinlaender G, Kornek G, Kletter K, Scheithauer W and Virgolini I (1996) Comparison of iodine-123-vasoactive intestinal peptide receptor scintigraphy and indium-11 l-CYT-103 immunoscintigraphy. J. Nucl. Med., 37, 1480–7. Raderer M, Kurtaran A, Leimer M, Angelberger P, Niederle B, Vierhapper H, Vorbeck F, Hejna MHL, Scheithauer W, Pidlich J and Virgolini I (2000) Value of peptide receptor scintigraphy using !23Ivasoactive intestinal peptide and '"in-DTPA-D-Phe1-octreotide in 194 carcinoid patients: Vienna University experience, 1993 to 1998.J. Clin. Oncol., 18, 1331–1336. Raderer M, Kurtaran A, Yang Q, Meghdadi S, Vorbeck F, Hejna M, Angelberger P, Kornek G, Pidlich J, Scheithauer W and Virgolini I (1998) Iodine-123-vasoactive intestinal peptide receptor scanning in patients with pancreatic cancer. J. Nucl. Med., 39, 1570–5.

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Rajodpadhye M, Overoye KL, Nguyen HM, Liu S, Edwards DS, Onthank D and Barrett JA (2000) The synthesis and evaluation of the 99mTc complexes of cyclic RGD-peptide antagonists of the integrin avpY Nucl. Med. Commun., 21, 577. Reubi JC (1995a) In vitro identification of vasoactive intestinal peptide receptors in human tumors: implications for tumor imaging. J. Nucl. Med., 36, 1846–1853. Reubi JC (1995b) Neuropeptide receptors in health and disease: The molecular basis for in vivo imaging. J. Nucl. Med., 36, 1825–1835. Reubi JC, Schaer J-C and Waser B (1997) Cholecystokinin (CCK)-A and CCK-B/gastrin receptors in human tumors. Cancer Research, 57, 1377–1386. Reubi JC, Schar J-C, Waser B, Wenger S, Heppeler A, Schmidt JS and Macke HR (2000) Affinity profiles for human somatostatin receptor subtypes SST1-SST4 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use. Eur. J. Nucl. Med., 27, 273–282. Reubi JC, Waser B, Schaer JC, Laederach U, Erion J, Srinivasan A, Schmidt MA and Bugaj JE (1998) Unsulfated DTPA- and DOTA-CCK analogs as specific high-affinity ligands for CCK-B receptorexpressing human and rat tissues in vitro and in vivo. Eur. J. Nucl. Med., 25, 481–490. Rogers BE, Rosenfeld ME, Khazaeli MB, Mikheeva G, Stackhouse MA, Liu T, Curiel DT and Buchsbaum DJ (1997) Localization of iodine-125-mIP-Des-Metl4-bombesin (7-13)NH2 in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer [see comments]. J. Nucl. Med., 38, 1221–9. Ruoslahti E (1991) Integrins. J. Clin. Invest., 87, 1–5. Safavy A, Khazaeli MB, Qin H and Buchsbaum DJ (1997) Synthesis of bombesin analogues for radiolabeling with rhenium-188. Cancer, 80, 2354–2359 Suppl. S. Schally AV (1988) Oncological applications of somatostatin analogues. Cancer Research, 48, 6977-6985. Schottelius M, Senekowitsch-Schmidtke R, Scheidhauer K, Kessler H, Schwaiger M and Wester HJ (2000) Glycated radioiodinated octreotides and octreotates with renal excretion and increased tumor uptake for SSTR scintigraphy and peptide receptor radionuclide therapy. J. Nucl. Med., 41, 40P. Schubiger PA, Allemann-Tannahill L, Egli A, Schibli R, Alberto R, Carrel-R6my N, Willmann M, Blauenstein B and Tourwe D (1999) Catabolism of neurotensins. Implications for the design of radiolabeling strategies. Q. J. Nucl. Med., 43, 155–8. Sefler AM, He JX, Sawyer TK, Holub KE, Omecinsky DO, Reily MD, Thanabal V, Akunne HC and Cody WL (1995) Design and structure-activity relationships of C-terminal cyclic neurotensin fragment analogues. J. Med. Chem., 38, 249–57. Seregni E, Chiti A and Bombardieri E (1998) Radionuclide imaging of neuroendocrine tumours: biological basis and diagnostic results. Eur. J. Nucl. Med., 25, 639–658. Shattil SJ (1995) Function and regulation of the (33 integrins in hemostasis and vascular biology. Thromb Haemost,74, 149-155. Siegrist W, Solca F, Stutz S, Guiffre L, Carrel S, Girard J and Eberle AN (1989) Characterization of receptors for alpha-melanocyte-stimulating hormone on human melanoma cells. Cancer Research, 49, 6352-6353.

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Smith-Jones P, Bischof C, Leimer M, Gludovacz D, Angelberger P, Pangeri T, Peck-Radosavljevic M, Hamilton G, Kaserer K, Steiner G, Schlagbauer-Wadl H, Macke H and Virgolini I (1998) 'MAURITIUS" a novel somatostatin analog for tumor diagnosis and therapy. J. Nucl. Med., 39, 223P. Smith-Jones PM, Stolz B, Brims C, Albert R, Reist HW, Fridrich R and Macke HR (1994) Galliom67/Gallium-68-[DFO]-octreotide - A potential radiopharmaceutical for PET imaging of somatostatin receptor-positive tumors: Synthesis and radiolabeling in vitro and preliminary in vivo studies. J. Nucl.Med.,35, 317–325. Spradau TW, Edwards WB, Anderson CJ, Welch MJ and Katzenellenbogen JA (1999) Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyltechnetium-labeled octreotide. Nucl. Med.Biol.,26, 1–7. Stroop SD, Nakamuta H, Kuestner RE, Moore EE and Epand RM (19%) Determinants for calcitonin analog interaction with the calcitonin receptor N-terminus and transmembrane-loop regions. Endocrinology, 137,4752–4756. Thakur ML, Marcus CS, Saeed S, Pallela V, Minami C, Diggles L, Le Pham H, Ahdoot R and Kalinowski EA (2000) 99mTc-labeled vasoactive intestinal peptide analog for rapid localization of tumors in humans. J. Nucl. Med., 41, 107–110. Tolmachev V, Bernhardt P, Forssell-Aronsson E and Lundqvist H (1999) [114mIn]-DTPA-D-Phe!-octreotide A candidate for radionuclide therapy. J. Labelled Cpd. Radiopharm.,42 Suppl 1, S726-S728. Vaidyanathan G and Zalutsky MR (1997) Fluorine-18-labeled [Nle4,D-Phe7]-cc-MSH, an a-melanocyte stimulating hormone analogue. Nucl Med Biol, 24, 171–178. Vallabhajosula S, Moyer BR, Lister-James J, McBride BJ, Lipszyc H, Lee H, Bastidas D and Dean RT (1996) Preclinical evaluation of technetium-99m-labeled somatostatin receptor-binding peptides. J. Nucl. Med., 37, 1016–1022. van Hagen PM, Breeman WAP, Reubi JC, Postema PTE, van den Anker-Lugienburg PJ, Kwekkeboom DJ, Laissue J, Waser B, Lamberts SWJ, Visser TJ and Krenning EP (1996) Visualization of the thymus by substance P scintigraphy in man. Eur. J. Nucl. Med., 23, 1508–1513. van Hagen PM, de Jong M, Breeman WA, Bernard HF, Schaar M, Van Gameren A, Srinivasan A, Schmidt M, Bugaj JE and Krenning EP (2000) 111in-labeled RGD analog for tumor imaging. J. Nucl. Med., 41,286P. Virgolini I (1997) Mack Forster Award Lecture - Receptor nuclear medicine: vasointestinal peptide and somatostatin receptor scintigraphy for diagnosis and treatment of tumour patients. Eur, J. Clin. Invest., 27, 793–800. Virgolini I, Kurtaran A, Leimer M, Kaserer K, Peck-Radosavljevic M, Angelberger P, Hubsch P, Dvorak M, Valent P and Niederle B (1998a) Location of a VIPoma by iodine-123-vasoactive intestinal peptide scintigraphy. J. Nucl. Med., 39, 1575–9. Virgolini I, Kurtaran A, Raderer M, Leimer M, Angelberger P, Havlik E, Li S, Scheithauer W, Niederle B, Valent P and Eichler H-G (1995) Vasoactive intestinal peptide receptor scintigraphy. J. Nucl. Med., 36, 1732–1739.

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Virgolini I, Leimer M, Handmaker H, Lastoria S, Bischof C, Muto P, Pangerl T, Gludovacz D, PeckRadosavljevic M, Lister-James J, Hamilton G, Kaserer K, Valent P and Dean R (1998b) Somatostatin receptor subtype specificity and in vivo binding of a novel tumor tracer, 99mTc-P829. Cancer Res., 58, 1850–1859. Virgolini I, Szilvasi I, Kurtaran M, Angelberger P, Raderer M, Havlik E, Vorbeck F, Bischof C, Leimer M, Domer G, Kletter K, Niederle B, Scheithauer W and Smith-Jones P (1998c) Indium-111-DOTAlanreotide: Biodistribution, safety and radiation absorbed dose in tumor patients. J. Nucl. Med., 39, 1928–1936. Waibel R, Alberto R, Willuda J, Finnem R, Schibli R, Stichelberger A, Egli A, Abram U, Mach J-P, Pluckthun A and Schubiger PA (1999) Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyI complex. Nature Biotechnology, 17, 897–901. Wray V, Nokihara K and Naruse S (1998) Solution structure comparison of the VIP/PACAP family of peptides by NMR spectroscopy. Ann. NYAcad. Sci., 865, 37–44.

24. RADIOLABELED ANTIBODIES FOR TUMOR IMAGING AND THERAPY MICHAEL R. ZALUTSKYA AND JASON S. LEWISB A

Duke University Medical Center, Box 3808, Durham, NC 27710, USA; BMallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Boulevard, Campus Box 8225, St. Louis, MO 63110, USA. INTRODUCTION Antibodies are proteins that have been generated in vivo in response to a particular molecular stimulus, known as the antigen. In principle, antibodies can be raised against antigenic substances associated with any aberrant cell population, even if the structure of the antigen is unknown. As knowledge concerning the molecular nature of antigens associated with tumors increased, antibodies that could bind with high affinity and specificity to cancer cells were developed. Antibodies reactive with cancer-associated antigens offer the exciting prospect of targeting of radionuclides to tumors by exploiting the molecular specificity of the antigen-antibody interaction. Early studies demonstrating the feasibility of this approach include those performed with polyclonal antibodies raised against carcinoembryonic antigen (CEA), isolated from human colorectal tumors (Gold & Freedman, 1965). Using radioiodinated, affinity-purified anti-CEA antibodies, preferential localization of radioiodine activity in human tumors implanted in hamsters was observed (Primus et al.,1977). Antibodies raised in goats and rabbits were instrumental to the early development of antibody-based radiopharmaceuticals; however, due to the polyclonal nature of these anti-sera, they consisted of a family of proteins with a wide range of affinity and specificity for their intended target antigen. This difficulty was circumvented by the introduction of monoclonal antibody technology by Kohler and Milstein (Kohler & Milstein, 1975). Monoclonal antibodies (MAbs) are proteins with a defined amino acid sequence and thus, have a unique antigen affinity and specificity. Compared with polyclonal antibodies, MAbs can be produced more conveniently and in a more reproducible fashion. Because of these advantages, the advent of hybridoma technology launched a major effort seeking to utilize radiolabeled MAbs as diagnostic and therapeutic radiopharmaceuticals. In this chapter, some of the factors influencing the selection of appropriate radionuclides and radiolabeling methodologies will be discussed. Variables to be considered include the nature of the antigenic target, the molecular form of the MAb, and the match between the characteristics of the radionuclide and the intended clinical application. With these considerations in mind, the current status of protein radiohalogenation and radiometal labeling approaches will be reviewed in the context of their application to the development of MAbbased diagnostic and therapeutic radiopharmaceuticals.

Handbook of Radiopltarmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons. Ltd

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PROPERTIES OF THE ANTIGENIC TARGET The characteristics of the antigenic target are of obvious importance in determining the selectivity and specificity of labeled MAb accumulation in tumors; however, they also are relevant to the selection of radionuclide and labeling method. A key property is the density of the antigen on the tumor cell surface or in the extracellular matrix. This is important to insure that adequate levels of radionuclide are concentrated in the tumor to permit efficient lesion detection or destruction. An antigen density of greater than 50,000 copies per cell is considered to be needed for efficient targeting with radiolabeled MAbs. Clearly, as the antigen density decreases, the specific activity (mCi/mg) that will be needed will increase. For this reason, many reactor-produced radionuclides are not ideal for labeling MAbs, particularly those reactive with antigens present in low concentration in tumors. The homogeneity of antigen expression also must be considered, particularly for radiotherapeutic applications. Although the ability to target MAb to tumors has been achieved even in circumstances where only 15% of tumor cells expressed the antigen (Partanen et al., 1992), the presence of a large population of antigen-negative cells will increase the need for radionuclides and labeling methods that can yield high-specific activity labeled MAbs. Furthermore, for radioimmunotherapy, heterogeneous antigen expression could lead to subpopulations of tumor cells that escape irradiation. This problem can be minimized through the use of radionuclides emitting radiation of relatively long range such as 90Y and l88Re that can kill a significant number of antigen-negative cells due to the multi-cellular range of their p-particles. The ideal labeling method for a particular MAb also is influenced by the biological fate of the target antigen after the labeled MAb-antigen complex is formed. Depending upon whether the antigen-antibody complex remains on the cell membrane, is shed into the circulation, or is internalized into the tumor cell, the labeled MAb will be exposed to different catabolic processes, necessitating different labeling strategies. For example, antigen-antibody complexes formed either within the tumor or in the circulation are degraded in the reticuloendothelial system. Retention of the radionuclide in these organs can be modulated by varying the nature of the linker between the labeled prosthetic group and the MAb. Perhaps the most problematic outcome from a labeling perspective is the internalization of the antigen-antibody complex because this process exposes the labeled MAb to intracellular catabolic processes, notably lysosomal proteolysis, which can lead to rapid loss of the radionuclide from the tumor cell (Press et al., 1990; Geissler et al., 1991). This is an important issue from a labeling perspective because many of the most promising antigenic targets for developing labeled MAb-based radiopharmaceuticals are rapidly internalized (Slamon et al., 1989; Press et al., 1994; Wikstrand et al., 1995). In general, labeling internalizing MAbs with radiometals results in higher retention of the radionuclide in tumor cells compared with those labeled by conventional radioiodination methods (Novak-Hofer et al, 1994). PROPERTIES OF THE ANTIBODY Unlike other radiopharmaceuticals discussed in this volume, radiolabeled MAbs are macromolecules. Although there are five classes of human immunoglobulins, the vast majority of antibodies of interest for radioimmunodiagnosis and radioimmunotherapy are IgG, the predominant immunoglobulin class found in the blood. A simplified structure of the IgG molecule is shown in Figure 1. This glycoprotein contains two identical

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polypeptide heavy (H) and light (L) chains linked together by disulfide bonds to form a (HL)2 dimer. The H chain has a molecular weight of about 50 kDa and contains five domains - VH, CH1, hinge, CH2 and CH3 - while the L chain has a molecular weight of about 25 kDa and is composed of the VL and CL domains.

Figure 1. MAbs and fragments.

The variable (V) domains control the binding of the IgG to its antigenic target, with the specificity of this interaction generally determined by only a few amino acids within these regions. On the other hand, the constant (C) domains are not involved in antigen recognition and the amino acid sequences of these regions are conserved for antibodies of a given subclass. There are four members of the human IgG family (IgG), IgG2, IgG3, IgG4) that differ in the amino acid sequences of their Fc region (CH2 and CH3 domains; Figure 1) and the nature of the hinge region linking the molecule (Turner, 1981). For IgG1, the H and L chains are composed of 450 and 212 amino acid residues, respectively. The hinge regions of the IgG classes vary with regard to their length as well as the number and position of disulfide bonds linking the HL monomers. Most of the MAbs used for radiolabeling are of murine origin because they were generated from immunized mice. However, patients frequently develop an immune response to these murine proteins, compromising their utility for

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certain applications. Two types of MAbs that have been developed to minimize this problem also are illustrated in Figure 1. Chimeric MAbs are constructed by combining murine V domains with the C domains from a human IgG molecule by genetic engineering (Morrison 1985). Humanized MAbs contain only those amino acid residues from the murine V domains involved in antigen recognition (Reichmann et al,, 1988). Although chimeric and humanized MAbs are less immunogenic, their residence time in the human circulation is much greater than their murine counterparts due to their human C domains. For this reason, the ideal radionuclide and labeling method for a particular MAb may vary, depending upon whether it is in murine, chimeric or humanized form. The stability of a MAb in vivo, and thus, its suitability for radiolabeling, appears to be influenced by the characteristics of its constant region. One parameter that may be important is the flexibility of the MAb hinge region because it can have an impact on the accessibility of the molecule to proteases (Tao & Morrison, 1989). The human IgG2 hinge region is more rigid than those of other human and murine IgG subclasses (Dangl et al., 1988). Several chimeric MAbs containing human IgG2 constant regions have been constructed and shown to have prolonged tissue residence times compared with their murine counterparts (Batra et al., 1994; He et al., 1994; Reist et al.,1997a). Comparison of the radiolabeled catabolites generated from a radioiodinated human IgG2 chimeric and an IgG2b murine anti-tenascin MAb indicated two important differences (Reist et al., 1997b). First, a significantly greater percentage of the radioiodine activity from the murine MAb was associated with a -75 kDa protein, presumably a Fab-Fc fragment created by the cleavage of interchain disulfide bonds. And second, loss of the label from the chimeric MAb due to deiodination was considerably lower than from the murine MAb. As a result of the latter, a radioiodination method which reduced dehalogenation improved the tumor accumulation of the murine MAb but did not have a significant effect on the chimeric MAb (Zalutsky et al.,1996). An additional consideration is whether an intact IgG or a lower molecular weight MAb fragment will be labeled. MAb fragments are of interest because their clearance from normal tissues and penetration into the tumor is more rapid than intact IgG, making it possible to utilize radionuclides with relatively short half-lives. Some of the enzymatically- and genetically-derived MAb fragments that have been utilized for targeting radionuclides to tumors also are illustrated in Figure 1. Both F(ab')2 fragments (~100 kDa) and single-chain Fv fragment (scFv) dimers (~52 kDa) have the advantage of having two antigen recognition sites, increasing their functional affinity, while Fab fragments (~50 kDa) and scFv molecules are monovalent. When selecting between a MAb and the various MAb fragments that are available, radiolabeling issues must be considered. As will be discussed in later sections, most MAb labeling strategies involve modification of either lysine or tyrosine residues on the protein. Substitution of the label into the antigen recognition site (i.e., the complementary determining regions (CDRs) within the V domains) of the MAb could destroy its antigen binding capacity. According to amino acid sequence data, an intact IgG contains an average of 54 tyrosines and 88 lysines per molecule (Kabat et al., 1991). If one assumes that all lysine and tyrosine are equally reactive, then the probability of labeling a lysine or tyrosine is low because the CDRs are such small portions of the IgG molecule. However, as the size of the molecule decreases, the probability of modifying the CDRs increases due to the fact that MAb fragments are created by exclusion of C region domains while retaining the CDRs. ScFv fragments constructed from just the VL and VH domains would be expected to be particularly vulnerable to loss of antigen binding capacity due to substitution of a radiolabel in the CDRs. The location and number of lysine and tyrosine

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in the V domains of two MAbs, TP-1 and TP-3, have been evaluated in order to develop optimized labeling strategies (Olafsen et al., 1995, 1996). Less than 25% of the lysine residues were in the CDRs for bom MAbs; in contrast, about 50% of the tyrosine residues were in the CDRs. Based on this information, a method involving lysine modification would be the more prudent approach for labeling TP-1 and TP-3 scFv fragments. It is important to bear in mind that the reactivity of amino acids within a protein actually can vary considerably due to differences in their microenvironments. Factors influencing the relative reactivity of a particular amino acid include proximity to hydrophobic domains or bulky residues such as tryptophan, and accessibility to solvent. The relative reactivity of amino acids on MAbs has not been investigated; however, studies with smaller proteins and peptides indicate that the iodination rate of individual tyrosine residues in these molecules can differ by factors of 50 to 100 (Dube et al., 1966; Seon et al., 1970). SELECTION OF THE RADIONUCLIDE Radionuclide selection criteria for labeled antibody imaging and therapy applications are different. For imaging, it is essential to achieve adequate contrast between tumor and normal tissue activity levels in a time frame compatible with the physical half-life of the radionuclide. Because the absolute magnitude and duration of tumor activity levels are less important, diagnostic applications benefit from the use of short half-life radionuclides, such as 18F or 64Cu for positron emission tomography (PET) and 99mTc or 123I for single photon emission computed tomography (SPECT), in tandem with rapidly clearing MAb fragments. For radioimmunotherapy, the goal is to maximize the radiation absorbed dose ratios between tumor and critical normal tissues; in general, intact IgG molecules labeled with longer half-life radionuclides are used. An additional consideration for labeled MAb therapy is to match the emission characteristics of the radionuclide to the size, location and geometry of a particular tumor. Irradiation of volumes with multicellular, cellular and subcellular dimensions could be accomplished with radionuclides emitting p-particles, a-particles and Auger electrons, respectively. For a more comprehensive discussion of radionuclide selection, as well as the current status of labeled MAb imaging and therapy, the reader is referred to a recent review (Bast et al., 2000). To date, MAbs and MAb fragments have been labeled with a wide variety of radiometals and radiohalogens, and both approaches offer certain advantages. The number of metallic radionuclides is considerably greater than the number of halogens, and radionuclide cost and availability is an important factor to be considered. For example, Cu and 131I emit p-particles of similar energy; however, 67Cu is available only sporadically and at a cost considerably higher than 131I. An additional consideration is whether appropriate radionuclides of the same 67

element are available so that both antibody imaging and radioimmunotherapy can be performed with chemically similar reagents. An additional factor in deciding between a halogen and metallic radionuclide relates to the catabolism of the label from the MAb and the tissue retention of labeled catabolites. With radioiodinated MAbs, dehalogenation can result in uptake of the label in the thyroid and the stomach, a problem that can be minimized through the administration of blocking doses of potassium iodide. Some metallic radionuclides such as 90Y localize in bone when released in ionic form and this can result in bone marrow toxicity. A number of studies have compared the tissue distribution and therapeutic efficacy of MAbs labeled with 131 I and 90Y (Buchsbaum et al., 1993; Sharkey et

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al., 1990, 1997), and the trends observed are typical of the different in vivo behavior of radiometal-labeled and radiohalogenated MAbs. In general, tumor accumulation of 90Y was higher than that of 131 I; however, tumor-tonormal tissue ratios were higher for 131I. As a result, 13lI-labeled MAbs were more effective than their 90Y-labeled counterparts when administered at equitoxic doses. However, it is important to note that these experiments did not use current state-of-the-art labeling approaches for either radionuclide. ANTIBODY LABELING Although methods developed for labeling one antibody generally can be utilized successfully for other antibodies, this is not always the case. Differences in isoelectric point can influence the pH range at which an antibody becomes dysfunctional, imposing varying restrictions on the pH at which MAb labeling procedures can be performed. Sensitivity to nonaqueous media and oxidants also may be different. Oxidizing reagents such as Nchlorosuccinimide and chloramine-T can oxidize cysteine, methionine and tryptophan residues (Shechter et al., 1975), and these modifications may be sufficient to alter the binding of the MAb to its antigenic target. For example, the anti-breast carcinoma MAb DF-3 cannot be radioiodinated using oxidant levels considerably lower than those successfully utilized with many other MAbs (Hayes et al. 1988). Thus, it is necessary to optimize the labeling conditions for each individual MAb of interest with the immunoreactivity of the labeled protein being the most important quality control measure. In the following sections, methodologies for labeling MAbs with radiohalogens and radiometals will be reviewed. Although radionuclides from a wide variety of other elements have been investigated for MAb labeling, this discussion will focus on procedures for labeling MAbs and MAb fragments with iodine, fluorine, and astatine, as well as copper, yttrium, technetium, indium, rhenium and bismuth. IODINE RADIONUCLIDES Iodine-131 (t1/2 = 8.08 d) was the first radionuclide used in patients for MAb imaging and therapy. Although its 364 keV y–ray is not ideal for diagnostic applications and complicates its use for treatment studies, 131I remains the most frequently utilized radionuclide for clinical radioimmunotherapy (Kairemo, 1996; Bast et al., 2000). On the other hand, 13.1 hr half-life 123I emits a 159 keV y–ray which is well suited for both planar and SPECT imaging, and 4.2 day half-life 124I can be used for PET imaging. Finally, 60.1 -day half-life 125I emits short-range Auger and Coster-Kronig electrons, and these are extremely cytotoxic if localized in close proximity to the cell nucleus. A major advantage of labeling MAbs with iodine radionuclides is the ability to image and treat a patient with radionuclides of the same element. This greatly facilitates the use of quantitative imaging to perform treatment planning and to define tumor and normal tissue dose-response relationships. Both SPECT and PET with 123I- and 124 I-labeled MAbs have been utilized for this purpose (Schold et al., 1993; Larson et al., 1992; Daghighian et al., 1993). Because of the availability of multiple iodine radionuclides that emit suitable y-rays for imaging, it is possible to perform dual-label experiments, for example, with 131 I-and 125I-labeled MAbs. In this way, different labeling methods and MAbs can be directly compared without having to be concerned about differences in tumor characteristics (size, antigen concentration, hemodynamics) which are likely to exist among different groups of animals or patients.

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DIRECT ELECTROPHILIC METHODS The standard method for the radioiodination of MAbs involves electrophilic substitution of the radioiodine directly on one of the constituent amino acids of the protein. In these procedures, an oxidizing agent is used to convert sodium iodide to a cationic species which then attacks the most electronegative sites on the MAb. At neutral pH, labeling of tyrosine residues ortho to the hydroxyl group predominates; however, substitution of the radioiodine on tryptophan, phenylalanine, histidine and cysteine also can occur (Rogoeczi, 1984), Although electrolytic and enzymatic approaches have also been developed, the most frequently utilized methods for generating the electrophilic radioiodinating species involve the oxidants lodogen (Fraker & Speck, 1978) or chloramine-T (Hunter & Greenwood, 1962). Direct iodination methods are simple, proceed in high yields and are adaptable to remote handling procedures for therapy-level labeling. On the other hand, a significant disadvantage of directly-labeled MAbs is that in most cases, they are extensively deiodinated in vivo to a much greater extent than in vitro. Deiodinases of varying specificity for the phenolic ring of iodotyrosines and both rings of iodothyronines are known to exist in several normal tissues including the thyroid, kidney and liver (Dumas, 1973; Leonard & Rosenbert, 1997; Smallridge et al., 1981), as well as in some tumors (Ong et al., 1986; Lee et al, 1989; Itagaki et al., 1990). The high level of dehalogenation of labeled MAbs in vivo probably reflects the action of these enzymes on the iodotyrosines created on the MAb because of their structural similarity to these thyroid hormones. N-SUCCINIMIDYL IODOBENZOATES Direct iodination methods as well as the Bolton-Hunter reagent (Bolton & Hunter, 1973) all substitute the halogen ortho to a hydroxyl group on an aromatic ring. The hydroxyl group facilitates electrophilic halogenation but also activates the ring toward hydrolytic as well as deiodinase-mediated dehalogenation. To circumvent this problem, protein radioiodination methods that do not require a hydroxyl group to activate the ring toward electrophilic halogenation have been developed. The most widely explored compound, N-succinimidyl 3-iodobenzoate (SIB), is illustrated in Figure 2. SIB is conceptually similar to the Bolton-Hunter reagent but a) lacks a hydroxyl group ortho to the iodination site and b) the two carbon spacer between the active ester and the aromatic ring was omitted to increase conjugation efficiency by minimizing competitive hydrolysis. Paired-label comparisons of MAbs labeled by the SIB and Bolton-Hunter methods have validated the rationale of these structural alterations (Vaidyanathan et al., 1990a). As shown in Figure 2, the tin precursor for SIB, N-succinimidyl 3-(tri-n-butyl)stannylbenzoate (STB) was synthesized in two steps from m-bromobenzoic acid (Zalutsky & Narula, 1987). The method uses tbutylhydroperoxide as the oxidant and is performed in CHCl3-acetic acid at a pH of 5-5.5. Yields of 80-90% are typical, and similar results have been reported for the synthesis of N-succinimidyl 4-iodobenzoate with Nchlorosuccinimide as the oxidant (Wilbur et al., 1989). Protein conjugation efficiency for SIB is dependent upon pH and protein concentration; coupling yields greater than 70% are obtained at pH 8.5 and above, and protein concentrations greater than 5 mg/ml.

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692

COOR

9OOH n-BuLi

N-hydroxysuccinimide

R3SnCI

DCC SnR3

CO-NI

R3Sn

MAb

Na131l

pH 8.5

TBHP, AcOH, CHCI3

SIB Figure 2. Shown is the method for producing N-succinimidyl 3-iodobenzoate (SIB) from the tin precursor N-succinimidyl 3(tri-n-butyl)stannylbenzoate (STB) in two steps from m-bromobenzoic acid. The method uses t-butylhydroperoxide as the oxidant and is performed in CHCl3-acetic acid at a pH of 5-5.5. Subsequent protein conjugation efficiency for SIB is dependent upon pH and protein concentration; coupling yields greater than 70% are obtained at pH 8.5 and above, and protein concentrations greater than 5 mg/ml.

The potential utility of SIB as a reagent for the radioiodination of MAbs was investigated in athymic mice bearing subcutaneous D-54 MG human glioma tumor xenografts. The anti-tenascin MAb 81C6 was radioiodinated via the SIB and Iodogen methods, and paired-label tissue distribution studies were performed (Zalutsky et al., 1989). SIB labeling decreased thyroid uptake of radioiodine by 40- to 100-fold, reflecting a lower level of deiodination for the SIB-labeled MAb. Furthermore, SIB increased tumor retention between two- and five-fold over the 8-day experimental period without resulting in a concomitant increase in normal tissue levels. In a subsequent study using 131I-labeled 81C6, SIB labeling significantly increased therapeutic efficacy in the same animal model compared with an equal dose of 81C6 labeled using the Iodogen method (Schuster et al., 1991). A number of SIB analogues have also been synthesized and evaluated as reagents for MAb radioiodination. These include SIB analogues with methoxy (Vaidyanathan & Zalutsky, 1990b) and methyl (Garg et al., 1993a) substituents, and the use of iodopyridine (Garg et al., 1991b, 1993b) and iodovinyl (Hadley & Wilbur, 1990) templates. A more comprehensive review of protein radioiodination methodologies can be found in a review by Wilbur (Wilbur, 1992). RADIOIODINATION METHODS FOR INTERNALIZING MABS Because of their rapid degradation by lysosomal proteolysis, MAbs that are internalized into tumor cells require particular labeling strategies. When internalizing MAbs labeled using conventional methods are bound to antigen-

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positive cells, cell-associated radioiodine rapidly decreases due to the escape of iodotyrosine from the cells (Geissler et al., 1991). Furthermore, labeling these MAbs by the SIB method does not increase cellular retention of radioiodine activity (Reist et al, 1996), Three approaches have been developed for residualizing radioiodine activity in tumor cells after MAb internalization: oliogosaccharide conjugates, positively-charged templates, and D-amino acid peptides. OLIGIOSACCHARIDE CONJUGATES Some oligosaccharides are resistant to lysosomal degradation, and this property has been exploited to trap radioiodine in lysosomes after the internalization of proteins labeled via oligosaccharide-tyramine conjugates (Thorpe et al., 1993). A widely utilized procedure involves conjugation of radioiodinated tyramine-cellobiose (TCB) to the MAb (Ali et al., 1988). TCB is synthesized by reductive animation, radioiodinated using chloramineT, and the labeled conjugate coupled to the MAb using cyanuric chloride. Labeling MAbs via the TCB method has been reported to significantly increase the retention of radioiodine in target cells following internalization compared with MAbs labeled using direct methods in both in vitro and in vivo models (Ali et al., 1990; Reist et al., 1995). Unfortunately, these investigations also noted several disadvantages with the TCB method. In one study evaluating several anti-epidermal growth factor receptor variant III (EGFRvIII) MAbs, TCB labeling resulted in lower immunoreactivity than seen with Iodogen and a higher fraction of aggregated species (Reist et al., 1995). In addition, MAbs labeled using TCB exhibited significantly higher retention of radioiodine activity in the liver and the spleen. This behavior likely reflects the potential for causing cross-linking due to the three replaceable chlorines in cyanuric chloride. Dilactitol-tyramine (DLT) also has been investigated for the radioiodination of internalizing MAbs. DLT is first labeled using lodogen, and then coupled to MAbs via the generation of aldehydes with galactose oxidase, followed by reductive amination using sodium cyanoborohydride (Thorpe et al., 1993). A drawback to this method is that the labeling efficiency is only 3 to 6% (Stein et al., 1995). Considerably higher retention of radioiodine activity in tumor xenografts has been observed for MAbs labeled using DLT, with a less pronounced increase also noted in liver, spleen and kidneys. Tumor regressions in athymic mice with Calu-3 adenomas of the lung were greater for MAb labeled via DLT compared with Iodogen (Stein et al., 1997); however, the low specific activity of DLT MAb preparations, due to poor coupling efficiency, was problematic. POSITIVELY CHARGED TEMPLATES Another strategy for labeling internalizing MAbs involves coupling a labeled molecule to the MAb that is positively charged at lysosomal pH. Because positively charged molecules such as neutral red and chloroquine are utilized as lysosomal markers, it was hypothesized that positively charged catabolites created during MAb proteolysis should also be trapped in the lysosome (Reist et al., 1996). N-succinimidyl 5-iodo-3pyridinecarboxylate (SIPC) was selected to test this hypothesis because its pyridine ring should yield labeled catabolites that are positively charged after MAb proteolysis. In addition, non-internalizing MAbs labeled using SIPC did not undergo dehalogenation in vivo and were not sequestered in normal tissues including the liver and spleen (Garg et al., 1991b),

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Internalization assays with the anti-EGFRvIII MAb L8A4 demonstrated that SIPC labeling increased cellular retention of radioiodine by up to 65% compared with MAb labeled using either SIB or Iodogen (Reist et al., 1996). HPLC analyses indicated that the radioiodide activity was retained within the cell primarily as the positively-charged iodonicotinic acid-lysine conjugate. When the tissue distribution of L8A4 labeled using SIPC and Iodogen was compared, MAb labeled by the SIPC method exhibited significantly higher tumor uptake over the first 72 hr after injection and higher tumo:normal tissue ratios. A subsequent study compared the targeting of MAb L8A4 labeled using the TCB and SIPC methods (Reist et al., 1997b). The conjugation yield for SIPC was 50-60%, about twice that obtainable with TCB. Tumor levels observed were similar for the two labeling methods, particularly at early time points; however, tumor-to-tissue ratios for kidneys, liver and spleen were 3 times higher for SIPC at later time points. D-AMINO ACID PEPTIDES Peptides composed of D-amino acids are resistant to degradation by lysosomal proteases (Ehrenreich & Conn, 1969). Recently, two conceptually different approaches involving D-amino acid peptides have been reported for the radioiodination of internalizing MAbs. In the first, D-Gly-Tyr-Lys was utilized to couple diethylenetriaminepentaacetic acid (DTPA) to a MAb with the objective of exploiting the fact that radiometalDTPA-MAb conjugates generally exhibit effective trapping of the metal within the tumor cell after internalization (Govindan et al., 1999). In vitro experiments demonstrated that when this peptide was radioiodinated on its tyrosine residue and coupled to internalizing MAbs, retention of radioiodine activity was up to 3-times higher than for the same MAbs labeled using chloramine-T The radioiodination of an internalizing MAb via a D-amino acid peptide containing several positively charged amino acids also has been investigated (Foulon et al., 2000). The prototypical peptide selected for evaluation was D-Lys-D-Arg-D-Tyr-D-Arg-D-Arg (D-KRYRR). Because the arginine side chain is the most basic (pKa 13.2) of the naturally occurring amino acids, 3 arginines were included to add multiple positive charges. A tyrosine was added to permit facile radioiodination and a lysine was included at the N-terminal to allow coupling of the peptide to the MAb. As shown in Figure 3, D-KRYRR was labeled using Iodogen and then activated by reaction with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC); meanwhile, free thiol groups were added to the MAb by reaction with 2-iminothiolane. Finally, the activated peptide was coupled to the MAb via a maleimido bond in 60% yield. Paired-label assays were performed to compare the properties of anti-EGFRvIII MAb L8A4 labeled using DKRYRR and Iodogen. Internalization assays with the U87AEGFR cell line indicated that internalized and total cell-associated activity for the 125I-labeled D-KRYRR-L8A4 conjugate were up to four and five times higher, respectively, than for L8A4 labeled with 131I using Iodogen. Paired-label comparisons in athymic mice with subcutaneous U87AEGFR xenografts demonstrated up to 5-fold higher tumor uptake for MAb labeled using DKRYRR.

RADIOLABELED ANTIBODIES FOR TUMOR IMAGING AND THERAPY

H N

AcOHN

1. Na*I, lodogen rt, 30 min. COOH

695

AcOHN *•

2. sSMCC rt, 30 min. NH2

OH

D-KRYRR

1. Coupling, rt, 45 min

2. Quenching with lodoacetamide rt, 20 min.

r

Figure 3. Schematic showing D-KRYRR labeling using Iodogen and then activation by reaction with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate; meanwhile, free thiol groups are added to the MAb by reaction with 2-iminothiolane. Finally, the activated peptide being coupled to the MAb via a maleimido bond.

LABELING MABS WITH ASTATINE-211 Alpha-particle emitters such as 211 At are of particular interest for therapeutic applications that can exploit their short range and high radiobiological effectiveness. Astatine-211 has a half-life of 7.2 hr that is better suited to the biokinetics of MAbs and MAb fragments than those of 61-min 212Bi and 47-min 213Bi, the other a-emitters that have received serious attention for radioimmunotherapy (RIT). Astatine-211 decays by a double-branched pathway with a-particle emission associated with each decay. In one branch (42%), a 5.87 MeV a-particle is emitted, and the other (58%) proceeds by electron capture to 500 msec 211Po, followed by the emission of 7.45 MeV a-particles. Because of the electron capture decay branch, polonium K x- rays of 77-92 keV, and these emissions permit the monitoring of 211At distributions in vivo by planar and SPECT imaging (Johnson et al. , 1995). In tissue, the range of•211 At a-particles is only 55 to 70 m, equivalent to only a few cell diameters. Because of their short range and high energy, a-particles are exquisitely cytotoxic. Indeed, the linear energy transfer (LET) for 211 At a-particles, ~100 keV7u.m, is about 500 times that of the p-particles of 90Y, and near the LET at which 211 the biologic effectiveness of radiation is highest (Hall, 1994). The high cytotoxicity of 211 At-labeled MAbs has been confirmed both in cell culture experiments (Larsen et al., 1996; 1998) and in animal models (Zalutsky et al., 1994). Indeed, cell killing has been achieved with as little as one a-particle traversal per cell.

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Astatine-211 is generally produced by bombarding natural bismuth metal targets with a-particles using the 209 Bi(a,2n)211At reaction. An incident a-particle beam energy of about 28.5 MeV is recommended in order to maximize yield while minimizing the production of 8.1 -hr210At. This impurity is problematic because it decays to 138-day 210Po, a potential source of bone marrow toxicity. The 211At is separated from the bismuth cyclotron target using dry distillation (Zalutsky & Narula, 1988). A signif1icant impediment to the development of 211Atlabeled MAbs is that only a few cyclotron facilities have sufficient a-particle beam energy and intensity to produce meaningful quantities of this radionuclide. This problem may be alleviated somewhat by the development of more efficient methodologies for high-level211 At production. Utilizing an internal cyclotron target specifically designed for 21l At irradiation (Larsen et al., 1996), up to 178 mCi of 21l At has been produced after 4-hr runs at beam currents of about 50 uA. It has been possible to run this target at a-particle beam currents of greater than 80 uA, so even higher levels of 211At should be readily obtainable with this internal target. ASTATINE-211 MAB LABELING CHEMISTRY Unlike the case with iodine, direct halogenation of constituent tyrosine residues on a protein is not a useful approach for 211At because of rapid dissociation of the carbon-astatine bond in oxidizing media (Aaij et al., 1975). More stable 21l At-labeled proteins were obtained with a two-step procedure involving the synthesis of p[211At]astatobenzoic acid from a diazonium salt precursor followed by conjugation to the protein via a mixed anhydride reaction (Friedman et al., 1977). However, this method had significant limitations including low conjugation efficiency and specific activity, as well as the formation of multiple by-products. A more successful approach has been to label MAbs using an analog of the SIB reagent, N-succinimidyl 3[211 At]astatobenzoate (SAB) (Zalutsky & Narula, 1988). Although reasonable yields could be obtained with the tri-n-butylstannyl precursor used in the iodination reaction, somewhat higher astatodestannylation efficiencies were possible with its trimethyl analog, presumably due to the larger bulk of the astatine atom compared with iodine. When the SAB method was utilized to label the anti-tenascin MAb 81C6, tumor levels were similar to those observed with co-administered [131I]SIB labeled MAb (Zalutsky et al., 1997). A clinical study has been initiated to evaluate the therapeutic potential of 211At-labeled chimeric 81C6, labeled by the SAB method, in patients with glioma (Zalutsky et al., 2000). Preliminary results suggest that this MAb is quite stable in vivo. LABELING MABS WITH FLUORINE-18 Combining radioimmunoscintigraphy with PET is a potentially valuable approach for increasing the sensitivity of lesion detection and providing quantitative information concerning MAb biokinetics that could permit patientspecific radio-immunotherapy planning. Fluorine-18 is an attractive radionuclide for PET because it is routinely produced at most PET centers and is available from commercial distributors. Unlike other positron emitters that have been used for labeling MAbs (124I and 64Cu), positron emission occurs with nearly all 18F decays, with no accompanying y-emission. These properties reduce the radiation dose received by the patient and enhance image quality. The half-life of 18F, 110 min, is not ideal for labeling intact MAbs and larger MAb fragments such as F(ab')2, but should be well suited to labeling scFv monomers or multimers.

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697

FLUOEINE-18 MAB LABELING CHEMISTRY The first two methods for labeling proteins with 18F were reported by Kilboum and co-workers (Kilbourn et al., 1987). Methyl 3-[l8F]fluoro-5-nitrobenzimidate

was produced by nucleophilic aromatic substitution of

18

[ F]fluoride for nitro in 3,5-dinitrobenzonitrile followed by reaction with excess sodium methoxide in anhydrous methanol. A drawback to this method is that a 1 hr reaction at 47°C was needed for protein coupling, which is not ideal because of the short half-life of 18F. In addition, the elevated reaction temperature could denature some MAbs, The other protein acylation agent described in this report was 4-[18F]fluorophenacyl bromide which was prepared in three steps beginning with fluoro-for-nitro exchange on 4-nitrobenzonitrile. Yields of greater than 75% were obtained labeling human serum albumin with a 1 hr reaction at 47°C. In a subsequent study, MAB BB5-G1, reactive with a human parathyroid surface protein antigen, was labeled with l8

18

F using 4-

18

f F]fluorophenacyl bromide and uptake of F in human parathyroid tissue xenografts in athymic mice was demonstrated (Otsuka et al., 1991). Another method for labeling MAbs with 18F utilizes N-succinimidyl l-[(4'-[18F]fluorobenzylamino)]suberate ([18F]SFBS) as the protein acylation agent (Garg et al., 1991 a). Several MAb Fab and F(ab')2 fragments have been labeled successfully using this four-step procedure. About 8-10 mCi of 18F labeled MAb fragments have been prepared from 100 mCi of [18F]fluoride in a synthesis time of 80-90 min. The properties of Mel-14 F(ab')2 fragment labeled via [18F]SFBS were compared to those when labeled using [125I]SIB (Garg et al. 1992), The affinity constant for 18F-labeled Mel -14 F(ab')2 was essentially identical to that for 125I-labeled F(ab')2, confirming the ability to label MAb fragments with 18F via [18F]SFBS with retention of immunoreactivity. There was an excellent correlation between the tumor uptake of 18F and 125I when the two labeled MAb fragments were studied in athymic mice bearing D-54 MG xenografts. Because of the potential for using PET to predict the dosimetry of a radioiodinated or 211At-labeled MAb, a radiofluorination agent analogous to SIB and SAB was developed (Vaidyanathan & Zalutsky, 1992a). Although these acylation agents are 3-halobenzoate esters, a para substituted compound was chosen for 18F because nucleophilic fluorination with the activating group in the meta position is problematic. The optimized method for the synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) involved three steps; [18F]fluoride for trimethylammomum substitution on 4-formyl-N, N, N-trimeAylanilinium triflate, oxidation to 4-[18F]fluorobenzoic acid, followed by conversion to [18F]SFB using N,N'-disuccinimidyl carbonate (Vaidyanathan & Zalutsky, 1994). Using O-(Nsuccinimidyl-N, N,N',N' -tetramethyluronium tetrafluoroborate, Wester et al., (1996) were able to obtain [18F]SFB in a radiochemical yield of 55% in a total synthesis time of only 35 min. The immunoreactivity, affinity and tumor localizing capacity of 18F-labeled Mel -14 F(ab')2 labeled via [18F]SFB were virtually identical to those observed when the MAb fragment was radioiodinated using SIB (Vaidyanathan & Zalutsky, 1992b). Another acylation agent that has been developed for labeling MAb fragments with

18

F is N-succinimidyl 4-

18

[ F](fluoromethyl)benzoate, prepared in a single step from N-succinimidyl-4-[(4-nitrobenzenesulfonyl) oxymethyljbenzoate (Lang & Eckelman, 1994). Reversed phase HPLC purification of this compound was required to maximize effective specific activity (Lang & Eckelman, 1997). The tissue distribution of a disulfide stabilized anti-Tac scFv labeled using N-succinimidyl 4-[18F](fluoromethyl)benzoate has been compared to that following radioiodination using the Iodogen method (Choi et al., 1995). Peak tumor levels for 18F were somewhat

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HANDBOOK OF RADIOPHARMACEUTICALS

lower that those reported for 125I. LABELING MABS WITH METAL RADIONUCLIDES PROPERTIES OF METAL RADIONUCLIDES There are a wide variety of gamma and positron emitting radiometals with very different decay characteristics and methods of production. As with non-metal radionuclides, the design of radiometal-based radiopharmaceuticals is based on factors such as the half-life of the radiometal, the mode of decay, and the cost and availability of the isotope. A recent review by Reichert et al., describes many of the issues involved with the design of metal-based radiopharmaceutical agents (Reichert et al., 1999). As with all radionuclides in diagnostic imaging, the half-life of the radionuclide must be long enough to carry out the desired chemistry to synthesize the radiopharmaceutical, but short enough to limit the dose to the patient. For diagnostic imaging in nuclear medicine, the majority of radionuclides decay primarily by gamma emission, since gamma scintigraphy is the most commonly used modality. Radionuclides used in PET decay by positron emission. The use of metal-based radiopharmaceuticals for therapeutic applications (alpha (a) or beta (p) emitters) is increasing, and many of the metal radionuclides also emit gammas or positrons, such as 111In and 64Cu, for applications in both therapy and imaging. Another important factor in choosing metal radionuclides for diagnostic imaging and therapy is their cost and availability. If the parent isotope is of relatively low cost, then even small medical centers can have a ready supply of the daughter radionuclide as needed. Radionuclide generators are considered ideal, because they consist of a longer-lived parent isotope that decays to a shorter-lived daughter radionuclide, and generator produced radionuclides are often less expensive than accelerator or reactor-produced radionuclides. The daughter can be easily separated from the parents by either ion exchange chromatography (the more common method) or solvent extraction. A few radiometals used in radiopharmaceuticals for gamma scintigraphy or PET are produced by a nuclear reactor. The most important of these is "Mo used to generate99mTc. Reactor-produced radionuclides are generally less expensive, because nuclear reactors have the capability to produce many isotopes at one time, compared to accelerators which only produce one isotope at a time. Designing metal complex based agents requires correlating aspects of the coordination chemistry with in vivo behavior. Redox properties, stability, stereochemistry, charge and lipophilicity of the metal complex are extremely important considerations. Another important factor to consider is complex stability; while thermodynamic stability of non-radioactive metal complexes can help predict in vivo behavior, it is often not indicative of in vivo stability. The following sections on metal-radiolabeled antibodies will primarily focus on the more common modes of attaching metals to biomolecules with particular attention made to agents that are currently approved for clinical use or are in the latter stages of clinical development. The radiolabeled agents described below are by no means exhaustive of the metals or antibodies that have been or are still under development for targeted radiotherapy applications.

BIFUNCTIONAL CHELATORS With radiometal-labeled MAbs, the radionuclide is often connected to the MAb via a bifunctional chelating agent (BFC), which consists of a chelator to complex the radiometal and a functional group for attachment to the biomolecule (Figure 4). To date, functional groups that form amide, thiourea, urea, Schiff base, or thioether

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699

linkages with amine or thiol groups on proteins and peptides have been described (Delmon-Moingeon et al., 1991; Gansow, 1991; Schubiger et al., 1996; Thakur, 1995). The first BFCs described were analogs of EDTA and DTPA (Hnatowich et al., 1983; Krejcarek & Tucker, 1977; Meares et al., 1976; Sundberg et al., 1974), Several improvements have been made to the originally developed BFCs, and they are described in a review article by Gansow (Gansow, 1991). Commonly used BFCs for radionuclides of copper, technetium, and rhenium are described in a review article by Schubiger and colleagues, (Schubiger et al., 1996). Two other thorough reviews discuss BFCs that have designed for isotopes of indium, technetium, yttrium, and rhenium (Hnatowich, 1994; Jurrison et al., 1993). The remainder of this section will focus on the most recent developments in metal-MAb conjugation chemistry. Metal Chelator

Radiometal

Covalent Linkage

F'igure 4. Bifunctional chelating agent (BFC), which consists of a chelator to complex the radiometal and a functional group for attachment to the biomolecule.

COPPER-64 AND COPPER-67 There is a diverse selection of diagnostic (60Cu, 61Cu, 62Cu and 64Cu) and therapeutic (64Cu and 67Cu) copper isotopes. The positron-emitting diagnostic nuclides have a wide range of half-life (10 min to 12.7 h), and are reactor, cyclotron or generator produced. Copper-67 is a therapeutic radionuclide that is currently produced on a high-energy accelerator. The reader is directed to a chapter in this volume that has been devoted to the chemistry of copper radionuclides and radiopharmaceutical products including an extensive section on copper labeled proteins and peptides (Anderson et al., 1994). Both intact MAbs and fragments have been labeled with 64Cu and 67

Cu for diagnostic imaging and RIT. The linking of the copper radionuclide to the MAb has been undertaken

employing a range of BFC's (Blower et al., 1996). For example, the bifunctional chelator of bromoacetamidobenzyl-1,4,8,11 -tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (BAT) with the linker 2iminothiolane (2IT) was employed to label MAbs Lym-1 (DeNardo et al., 1991) and 1 A3 (Philpott et al., 1995) with copper, and the macrocyclic chelator 4-[(l ,4,8,11 -tetraazacyclotetradec-1 -yl)methyl]benzoic acid (CPTA) was used to label MAb 35 (Smith-Jones et al., 1991), chCE7 F(ab')2 (Zimmerman et al., 1999) and MAb SEN7 (and its F(ab')2 fragments) (Smith et al, 1994) with 67Cu.

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YTTRIUM-90 Yttrium-90 (t1/2 = 64 h) has utility in therapy applications. Its mean P- energy of 0.9 MeV with a maximum energy of 2.27 MeV and a maximum particle range of about 11 mm in tissue, makes it an appropriate radionuclide for large tumor burdens. Because 90Y does not have y-emissions, imaging is difficult and can only be performed with the analogous 111In-labeled MAb. Potential differences in uptake and clearance of the two metals could give rise to inaccurate dosimetry estimates. Yttrium-90 is commercially and economically available in high specific activity. In aqueous solution, the most prevalent species is Y(III), and therefore, many of the same chelates that complex In(III) in a stable configuration have been utilized with Y(III). The majority of MAb studies using yttrium radioisotopes involve labeling the biomolecule through a BFC, generally, derivatives of diethylenetetraaminepentaacetic acid (DTPA) and l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA). It has been shown that 90Y dissociates from DTPA in vivo and accumulates in the bone (Hnatowich et al., 1988). For this reason, more stable MAb-radiometal conjugates modified with derivatives of DTPA have been developed. A frequently used type of bifunctional DTPA compound for MAb conjugation are the pisothiocyanotobenzyl derivatives (Brechbiel et al., 1986; Westerberg et al., 1989). A human therapy trial with 90Y employed the derivative p-isothiocyanotobenzyl-DTPA conjugate of the MAb cT84.66 (Westerberg et al., 1989). The macrocyclic chelator DOTA has replaced DTPA in many instances since it forms stable yttrium complexes of very high kinetic stability (Deshpande et al., 1990). Although a number of methods exist for attaching DOTA to proteins, one of the more simple methods employs a water-soluble chemical reagent (Lewis et al., 1994). This procedure involves the activation of a single carboxyl group on DOTA with N-hydroxysulfosuccinimide (sulfoNHS). The sulfo-NHS ester of DOTA is prepared in a single step using l-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC). The DOTA-MAb conjugates are then prepared by adding the active ester reaction mixture to the proteins at pH 8.5-9.0. Generally, when labeled with 90Y in acetate or Tris buffer, the immunoreactivity of the labeled MAb is maintained. The number of reports relating to conventional RIT with 90Y and intact MAbs has decreased due to the inability to deliver sufficient radiation dose to tumors. To overcome some of the obstacles associated with conventional RIT, there have been an increasing number of reports in the area of pretargeted RIT (Figure 5). Pretargeting involves administration of an antigen specific MAb which is covalently linked to a molecule having a high-affinity noncovalent binding site for a small rapidly excreted effector molecule. First, the unlabeled MAb-binder is given, often followed by a clearing agent. This clearing agent, while leaving the MAb on the tumor, will remove any unbound MAb from the circulation, significantly improving the tumor/blood ratio. The radiolabeled effector molecule is injected soon after the clearing agent. This pre-targeting strategy allows the radiolabeled small molecule to bind to the tumor with excretion of residual radioactivity. An example of a three-step pre-targeting strategy for RIT involves the use of the murine IgG2b MAb NR-LU-10 that recognizes a 40-kDa epithelial antigen known as Ep-CAM (Okabe et al., 1984). The NR-LU-10 fragment is located on numerous tumors, including lung (small cell and non-small cell), kidney and prostrate (Breitz et al., 1997; Straka at al., 2000). Step 1 is the administration of a streptavidin-conjugated MAb NR-LU-10 moiety (SA-NR-LU-10). This was followed by the administration of a sugar-based clearing agent, coupled to a biotin-human serum albumin (biotin-HSA) conjugate. The third step was the administration of a DOTA-biotin conjugate labeled with 90Y. Pretargeted RIT with SA-NRLU-10, biotinylated galactosyl-HSA and 90Y-DOTA-biotin showed extremely promising results in nude mice carrying human tumor xenografts, with 100% cures in mice administered 800 uCi 90Y-DOTA-biotin (Axworthy et

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al, 2000). The results from human clinical trials were not quite as successful, however. In a Phase II study of 25 patients with metastatic colon cancer, the same pretargeting strategy was used, and the overall response rate was 8%, and 16% of patients showed stable disease (Knox et al., 2000). Hematological and gastrointestinal (GI) toxicity was observed, since MAb NR-LU-10 reacts with normal GI epithelium. The toxicity has limited the amount which could be administered, and hence, likely limited the efficacy of SA-NR-LU-l0/90Y-DOTA-biotin.

1 -2 days SA

mAb-Streptavidin (MAb-SA) - tumor targeting entity

Clearing agent

- galactosylated protein or polymer binds to MAb-SA in blood and clears it from the body via the liver

SA

1–10 hours

Biotin-linker-chelate radiometal - binds to MAb-SA remaining in body; any unbound is rapidly excreted

Figure 5. Schematic showing radiotherapy by pretargeting. TECHNETIUM-99M 99m

Tc (t1/2 - 6,0 h) is the most widely used radionuclide for diagnostic imaging, accounting for over 80% of scans

performed in the US. Technetium radiopharmaceuticals are used routinely for the imaging of diseased tissues in the human body, e.g., heart, brain, kidney and bone. Technetium coordination chemistry is diverse as a consequence of the range of available oxidation states (-1 to +7), a wide number of coordination geometries, and its ability to bind to a large range of donor ligands to fulfill its coordination requirements. The reader is directed to a chapter in this volume discussing the most recent developments in technetium chemistry (Jones). Eckelman has published an exhaustive review of 99mTc radiopharmaceuticals that includes a fairly complete section on BFCs for labeling peptides and proteins with 99mTc (Eckelman, 1995). Alternative methods for labeling proteins with 99m

Tc involve reducing intramolecular disulfide bonds to generate thiol groups which have high affinity for Tc(V).

Several review articles discuss the various direct protein labeling methods for 99mTc and rhenium isotopes (Fritzberg, 1987; Eckelman & Steigman, 1991 ; Griffiths etal, 1992; Hnatowich, 1994). More recently, methods for direct labeling of 99mTc- and l88Re- peptides have been reported (Thakur et al, 1997; Thakur et al, 1996). Currently, 99mTc-labeled carcinoembryonic antigen (CEA-Scan; Immunomedics, Inc., Morris Plains, NJ) and 99

Tc-nofetumomab merpentan (Verluma™;Boehringer Ingelheim Pharma KG., Germany) are approved for human

use in the US. CEA-Scan is composed of a murine monoclonal Fab' fragment generated from IMMU-4, a murine IgG1 MAb, against the CEA surface antigen found on colorectal carcinomas (Goldenberg et al., 1997a; Erb & Nabi, 2000). It

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is approved for use in the US, Europe and Canada as an imaging agent for detection or recurrent or metastatic colorectal carcinoma. The technetium is labeled to the MAb by reacting with pendant sulfhydryl groups resulting from the reduction of disulfide bonds within the protein. Using one or more pendant groups on the protein as endogenous ligands and using an exogenous ligand, mere is no special need for a bifunctional chelator. The direct method of labeling does have some universal limitations, since severe denaturation and subsequent loss of immunoreactivity of the antibody is possible in the presence of the reducing agents required to reduce the metal's oxidation state. CEA-scan has a short biological half-life and rapid blood clearance, which results in improved target-to-background ratios. Pharmacokinetic studies revealed blood levels of 63%, 23%, and 7% of the injected dose at 1,5 and 24 hours after infusion, respectively. Verluma™ consists of a Fab' fragment of the pancarcinoma murine IgG2b antibody NR-LU-10. The BFC employed used to attach the technetium metal to the antibody is a phenthiolate ligand, 2,3,5,6-tetrafluorophenyl-4,5-bis-S-(l -ethoxyethyl)-thioacetoamidopentanoate following reduction of the Tc by a stannous gluconate complex. INDIUM-111 Indium-111 (t1/2 = 62 h), the most widely used indium radionuclide, is cyclotron produced, decays by electron capture (EC) with subsequent emission of gamma photons of 173 keV and 247 keV (89 and 94% abundance, respectively), and is widely used in gamma scintigraphy. Indium-111 was first evaluated in vivo in the late 1960's and since then, there have been a number of reviews which cover 111 In radiopharmaceuticals (Welch & Welch, 1975; Thakur, 1977; Anderson etal., 1994). Many of the 111In-labeled imaging agents that were developed in the 1970's are still used today as diagnostic agents for renal and brain imaging, imaging flow changes and leakage of cerebrospinal fluid and for imaging infection. The majority of new 111ln radiopharmaceuticals are biomolecules such as proteins and peptides. Indium is only stable in the +3 oxidation state in aqueous solution. Many nitrogen, oxygen and sulfur containing ligands bind In(III) with high stability. Indium(III) is considered intermediate with respect to the Hard Acid/Hard Base (HAHB) theory, preferring neutral nitrogen and negative sulfur donor atoms. Indium(III) also hydrolyzes easily, forming insoluble hydroxides at pH > 3.4, and forms very strong complexes with transferrin. A chapter in this volume discusses in greater detail indium (and gallium) chemistry and how it relates to pharmaceutical production (Thakur, 1977). In more recent years, there have been renewed efforts toward the development of a more stable chelate for In(III). This is due in part to the advent of 111In-labeled MAbs and peptides using bifunctional chelating agents. Currently, indium-111 labeled DTPA-B72.3 (OncoScint™; Cytogen, Princeton, NJ), DTPA-IgG anti-myosin (Myoscint™; Centocor B.V., Leiden, The Netherlands) and DTPA-7E11.C5.3 (ProstaScint™; Cytogen, Princeton, NJ) are approved for clinical use in the US. The MAb B72.3 in OncoScint™ specifically targets the cell surface mucin-like glycoprotein antigen TAG-72 that is found on approximately 83% of colorectal carcinomas and 97% of ovarian carcinomas (Kruglyak et al., 1997; Goldenburg, 1997b; Pinkas et al., 1999). Tumor detection is significantly higher in humans with positive TAG-72 serum levels and when the percentage of tumor cells expressing the antigen is 40% or higher. The monoclonal antibody fragment in Myoscint™ binds irreversibly to exposed myosin filaments of damaged myocytes and is used as an agent to diagnose myocardial injury (Johnson at al., 1989). The antibody is targeted against the heavy chain of human cardiac myosin, a large intracellular protein in cardiac muscle cells that is exposed only when the integrity of the cell membrane is irreversibly disrupted, since myosin is expressed exclusively in the intracellular compartment. ProstaScint™ is an

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murine monoclonal antibody (MAb 7E11-C5.3) that targets the intracellular epitope of prostrate-specific membrane antigen (PSMA). PSMA is a well-characterized cell surface antigen expressed by virtually all prostrate cancers. The MAb is attached to the chelator glycyl-tyrosyMMe-diethylenetriaminepentaacetic acid)-lysine (GYK-DTPA) to form the immunoconjugate for subsequent labeling with 111In. The drug is approved by the FDA as a diagnostic imaging agent in newly diagnosed patients with biopsy-proven prostate cancer and for use in postprostatectomy patients in whom there is a high suspicion that the cancer has recurred (Petronis et al., 1998; Texter & Neal, 1998; Elgamal et al, 1998). There is a high degree of tumor binding to all prostate cancers tested in vitro with a high degree of specificity. There is no significant binding to other cancers or normal tissues, with the exception of mild binding to benign prostatic hypertrophic and normal prostate tissue. Reliable SPECT imaging of tumors of a size 0.5 g and above can be obtained. Although ProstaScint™ has been successfully targeted in vivo, it binds to an intracellular epitope of PSMA and the MAB 7E11 is not internalized. Recently, murine MAbs J415, J533 and J591 were radiolabeled with 13I I and evaluated in competitive and saturation binding studies (Smith-Jones et al., 2000). In the same study, J415 and J591 were also conjugated to DOTA (up to five DOTA chelates bound to either MAb) and labeled with 111In without compromising immunoreactivity. A comparison of the cellular uptake and metabolic processing of the 131I- and 111In-labeled antibodies showed a rapid elimination of I31

I from the cell and a high retention of !11In. Both 111In-DOTA-J415 and 111In-DOTA-J591 had a similar nanomolar affinity to PSMA compared with ProstaScint™ but were far more readily bound and internalized. This indicated that both DOTA-J415 and DOTA-J591 are promising MAbs for the targeting of viable PSMAexpressing tissue with diagnostic and therapeutic metallic radionuclides. RHENIUM- 186 AND RHENIUM- 188 Two rhenium radionuclides have been evaluated for RTF. Rhenium-1 86 (t!/2 = 3.7 d) has abundant intermediateenergy p emissions of 1.07 MeV (71%) and 0.94 MeV (21%). Rhenium-186 is produced by the 185Re(n,y) reaction in a nuclear reactor, resulting in low specific activity due to the presence of carrier from the target. Rhenium-1 88 (t1/2 = 16.9 h) can be obtained at reasonable cost from a 188W/188Re radionuclide generator in high specific activity. It decays by p" emission with energies (Emax P" = 2.12 MeV) similar to 90Y. The y-emission following P decay in both 186Re (E7 = 137 keV; 21%) and 188Re (E7 = 155 keV; 15%) allows imaging for estimating dosimetry of the radiopharmaceutical used for a therapeutic application. Rhenium is the group VII congener of technetium and the chemical similarity between the two elements stems from the 'lanthanide contraction' observed for second and third row transition metals. As a consequence of this similarity, therapeutic rhenium radiopharmaceuticals have been developed based on 99mTc-imaging agents. The coordination compounds of the two elements are similar in terms of size, geometries, dipole moments and lipophilicity. However, on comparison with their analogous rhenium agents, technetium complexes are more easily reduced. Direct labeling methods often employed with Tc are therefore not ideal for Re because of the requirement for stronger reducing agents. For example, the stability of 188Re- and 99nTc-labeled MAb anti-NCA95 determined in vitro showed the 99mTc-MAb was 100 % intact after 24 h, whereas the !88Re-labeled MAb showed 30% dissociation of the radiometal from the antibody (Kotzerke et al. , 2000). Although direct labeling is not ideal, it can still be partially effective. For example, in a 1998 clinical study (Juweid et al., 1998), 188Re-MN14 IgG, a MAb directed against gastrointestinal cancer, was used with partial success in humans with

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histologically proven, CEA-producing, gastrointestinal cancer. The direct labeling of the MAb ultimately resulted in relatively rapid blood pool clearance, due in part, to the relative in vivo instability of the radiolabeled MAb. To overcome the inherent problems with indirect labeling methods, BFCs for Re have been developed using ligands based on the diamidodithiol or diaminodithiol systems (N2S2) (Fritzberg, 1987), N2S4 systems (Najafi et al., 1992) and the triaminothiol N3S frame work (Visser et al, 1993; Goldrosen et al., 1990). The majority of BFC systems for Re-MAbs are currently based on the N3S system. Among them, a pre-chelating labeling method using S-benzoyl-mercaptoacetyltriglycine (MAG3) is widely used, since it affords high in vivo stability and maintains high specific activity of the Re-labeled MAb (Visser et al., 1993). For some applications, mercaptoacetyldiglycyl-yaminobutyric acid (MAG2-GABA) is used to provide a spacer between the metal and biomolecule, which in some instances can be advantageous by reducing steric hindrance, improving labeling and increasing binding affinities. An impressive overview of 186/l88Re-labled biomolecules is contained within a review by Volkert and Hoffman (Volkert & Hoffman, 1999).

BISMUTH-212 AND BISMUTH-213 Non-radioactive bismuth compounds have relevance in both biology and medicine as shown by an exhaustive review by Briand and Burford (Briand & Burford, 1999). Two alpha particle-emitting isotopes of bismuth are currently available, 2l2 Bi and 2l3 Bi. Bismuth-212 (t1/2 = 60.6 min) decays to both 208T1 and 2l2 Po with an average a-particle energy of 7.8 MeV, while both 208T1 and 2l2 Po decay to 208Pb with each pathway resulting in the emission of an a-particle. The 2l2 Bi decay is accompanied by the emission of high-energy (2.6 MeV) photons requiring heavy shielding. A 224Ra/212Bi generator has led to the wider availability of the isotope, allowing for convenient production of bismuth labeled compounds. Bismuth-213 is similar in decay characteristics to 2l2 Bi, decaying by a (6 MeV, 2 %), p (98 %) and y (17 %) emissions to the ultra-short-lived a-emitter 2l3Po (t1/2 = 4.2 us; 8 MeV, 98 %). In humans, the 440-keV photon emission enabled imaging of 213Bi-labeled HuM195 in leukemia patients to derive pharmacokinetics and dosimetry. MAb HuM 195 is a humanized monoclonal antibody reactive with the cell surface antigen CD33, which specifically targets and kills myeloid leukemia cells (Sgourous et al., 1999). Produced by the decay of 225Ac, the 46-min half-life 2l3 Bi is available through a generator system that is capable of producing over 25 mCi of high-grade 2l3 Bi. Bismuth radionuclides can be readily attached to antibodies through BFCs such as functionalized DTPA derivatives. Bismuth isotopes do not generally bind strongly to cyclic-DTPA and although some derivatives were stable in vitro, this stability was not maintained in vivo. One derivative that has proven to be very stable in vivo is rranj-cyclohexyldiethylenetriamine pentaacetic acid (CHX-A-DTPA). CHX-A-DTPA has been used to rapidly chelate 213Bi, with the reaction completing in 1 -20 min with a radiochemical yield of 90% (Brechbiel et al., 1986). This BFC-MAb maintained high immunoreactivity and was labeled with 2l3 Bi in high specific activity. It is currently in use in pre-clinical and clinical trials with the anti-CD33 MAb HuM195 (Sgourous et al., 1997; Nikula et al., 1999).

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SUMMARY Significant progress has been made in the field of targeted radiotherapy with radiometals over the last decade. All of these agents have often been developed with a goal of decreasing toxicity while increasing affinity and delivery to the tumors. Targeting properties of these antibodies can now be safeguarded by careful choice of the BFC in order not to disrupt antigen binding efficiencies, while allowing stable chelation of the metal. In this respect, perhaps the area of most clinical and commercial interest at the moment is in the development of novel pretargeting methods for the treatment of cancer. This elegant method of improving target-tissue uptake results in a significant increase in the delivery of the radioisotope with a concomitant decrease in toxicity to healthy unaffected organs. Clinical trials are now underway to ascertain the true potential of this mode of treatment. In some instances, immunoreactivity has been enhanced by the use of carrier molecules or by the attachment of well-designed BFCs. The situation exists where a BFC-chelator-MAb conjugate could be developed for potential use with a myriad of radiometals. For example, the BFCs based on DOTA are known to produce highly kinetically stable metal complexes with, amongst others, copper, yttrium, indium, lutetium, samarium and gallium. This permits the same MAb conjugate to be labeled with different radiometals. This would allow for a "cocktail" approach, where the choice of radiometal would be tailored to an individual treatment regimen depending on the size of the tumors targeted, i.e., 90Y for large tumor burdens and 64Cu for metastatic disease. CONCLUSION This chapter has outlined the factors influencing the selection of appropriate radionuclides and radiolabeling methodologies for antibodies. The current status of protein radiohalogenation and radiometal labeling approaches has been reviewed in the context of their application to the development of MAb-based diagnostic and therapeutic radiopharmaceuticals. This review is by no means exhaustive and the reader is directed to the wealth of literature available on MAb technology as it relates to both imaging and radiotherapy. New and innovative advances are constantly being presented in order to improve the current status of this technology. For example, new labeling methods and new molecular biological technology for construct generation ensures that this area remains prominent in diagnostic and therapeutic medicine. Moreover, advances in radionuclide production techniques expand the utility of radiolabeled MAbs to the clinician; PET imaging/therapy pairs (e.g., 86Y/90Y, 64Cu/67Cu, 64

Cu/64Cu, 94mTc/188/186Re, 124I/131I) are of great interest. The use of these pairings in the radiotherapy arsenal is

particularly inventive and allows for the accurate determination of therapeutic response, dosimetry and pharmacokinetics with compounds that previously were difficult to image or monitor.

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Garg S, Garg PK and Zalutsky MR (1991b) N-Succinimidyl-5-(trialkylstannyl)-3-pyridine-carboxylates: a new class of reagents for protein radioiodination. Bioconj. Chem., 2, 50–56. Garg S, Garg PK, Zhao X-G, Friedman HS, Bigner DD and Zalutsky MR (1993b) Radioiodination of a monoclonal antibody using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Nud. Med. Biol., 20, 835–842. Geissler F, Anderson SK and Press O (1991) Intracellular catabolism of radiolabeled anti-CD3 antibodies by leukemic T cells. Cell Immunology, 137, 96–110. Gold P and Freedman SO (1965) Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J. Exp. Med., 121, 439-462. Goldenberg DM, Juweid M, Dunn RM and Sharkey RM (1997a) Cancer imaging with radiolabeled antibodies: New advances with technetium-99m-labeled monoclonal antibodies Fab' fragments, especially CEA-Scan* and prospects for therapy. J. Nud. Med. Tech., 25, 18–23 Goldenberg DM. (1997b) Perspectives on oncologic imaging with radiolabeled antibodies. Cancer, 80,2431–5. Goldrosen MH, Biddle WC, Pancook J, Bakshi S, Vanderheyden J-L, Fritzberg AR, Morgan AC and Foon KA (1990) Biodistribution, pharmacokinetic, and imaging studies with 186Re-labeled NR-LU-10 whole antibody in LS174T colonic tumor-bearing mice. Cancer Research, 50, 7979-7989. Govindan SV, Mattes MJ, Stein R, McBride BJ, Karacay H, Goldenberg DM, Hansen HJ and Griffiths GL (1999) Labeling of monoclonal antibodies with diethylenetriaminepentaacetic acid-appended radioiodinated peptides containing D-amino acids. Bioconj. Chem., 10, 231 –240. Griffiths GL, Goldenberg DM, Jones AL and Hansen HJ. (1992) Radiolabeling of monoclonal antibodies and fragments with technetium and rhenium. Bioconj. Chem., 3,91–99. Hadley SW and Wilbur DS (1990) Evaluation of iodovinyl antibody conjugates: comparison with a piodobenzoyl conjugate and direct radioiodination. Bioconj. Chem., 2, 50–56. Hall EJ (1994) Radiobiology for the Radiologist. Philadelphia, Lippincott. Hayes DF, Noska M, Kufe D, Zalutsky M (1988) Effect of radioiodination on the binding of monoclonal antibody DF3 to breast carcinoma cells. Nucl. Med. & Biol., 15, 235–241. He X, Archer GE, Wikstrand CJ, Morrison SL, Zalutsky MR, Bigner DD and Batra SK (1994) Generation and characterization of a mouse/human chimeric antibody directed against extracellular matrix protein tenascin.y, Neuroimmu., 52, 127–137. Hnatowich DJ (1994). The in vivo use of metallic radioisotpes in cancer detection and imaging. In Metal Comp. Can. Ther., Flicker, S.P. (ed) pp. 215-247. Chapman & Hall: London. Hnatowich DJ, Layne WW and Childs RL. (1983). Radioactive labeling of antibody: a simple and efficient method. Science, 220,613–615. Hnatowich DJ, Chinol M and Siebecker DA (1988) Patient biodistribution of intraperitoneally administered yttrium-90-labeled antibody. J. Nucl. Med., 29, 1428–1434. Hunter WM and Greenwood FC (1962) Preparation iodine-131 labelled human growth hormone of high specific activity. Nature, 194, 495-496. Itagaki Y, Yoshida K, Ikeda H, Kaise K, Kaise N, Yamamoto M, Sakurada T and Yoshinaga K (1990) Thyroxine 5-deiodinase in human anterior pituitary tumors. J. Clin. Endocrin. Metab., 71, 340–344. Johnson EL, Turkington TG, Jaszczak RJ, Garg P, Vaidyanathan G, Coleman RE and Zalutsky MR (1995) Quantitation of 211 At in small volumes for evaluation of targeted radiotherapy in animal models. Nucl. Med. & Biol., 22, 45-54.

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25. RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE) JOHN A. KATZENELLENBOGEN

Department of Chemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801, U.SA. INTRODUCTION

The Role of Tumor Receptor Imaging in the Management of Hormone-Responsive Cancer The design and development of agents to image hormone receptors has been an active and growing area of radiopharmaceutical chemistry and one that is beginning to have considerable medical impact. Through receptor-based imaging it is often possible to identify pathological conditions, stage disease, and monitor the effectiveness of therapy at an early stage. Although in the past neuroreceptors were the principal focus of receptor-based imaging agents, more recently, applications of receptor-based imaging in cancer have grown enormously. Many tumors that arise in hormone-sensitive tissues have significant levels of receptors that bind their hormonal ligands with high affinity. When properly designed, radiolabeled analogs of these hormones can be used to provide images of these tumors that can assist in the identification of primary and metastatic tumors and can provide valuable information about the responsiveness of these tumors to hormone therapy.

Hormone therapy for many cancers is often effective, because hormones that regulate the development and function of various normal tissues usually continue to play a growth regulatory role in tumors that arise from these tissues. This is the case for several nuclear hormone receptor systems that act at the level of transcription to regulate tissue growth. For example, in certain breast tumors, estrogens stimulate tumor growth, and hormone therapy that blocks estrogen action, either by treatment with antiestrogens or with aromatase inhibitors, can cause marked tumor regression (Allegra et al., 1979; Clark & Mcguire, 1983; Merkel & Osborne, 1989; Santen & Harvey, 1999; Santen et al., 1990). The same is true to some degree with androgens in prostate cancer (Petrylak, 1999), glucocorticoids in lymphoid cancers, and peroxisome proliferator-activated receptor ligands in certain other cancers (Sarraf et al., 1998). In general, when it is effective, hormone therapy is preferred over radiation or chemotherapy because of its lower morbidity. However, not all tumors that arise in normally hormone-sensitive tissues do respond to hormone therapy, and sometimes the response to hormone therapy is only of limited duration.

Besides nuclear receptors, there are other receptors (sigma receptors) that bind small molecule ligands and are amenable to imaging that might also provide valuable information for cancer detection and monitoring therapy. Sigma receptors are membrane receptors that are found in several neuronal tissues and in various endocrine, immune, and other peripheral tissues (Wolfe & De Souza, 1994). Although their biological function is not known, sigma receptors are also of great interest in cancer, because they are found at high Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wilev & Sons, Ltd

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levels in certain tumor cells, especially those in a high rate of proliferation (Al-Nabulsi et aL, 1999; Bowen, 2000; John et aL, 1997; Mach et al, 1997; Vilner et al, 1995; Wheeler et al, 2000). Although the endogenous ligands for the sigma receptors are not known, there are a number of synthetic compounds that bind to these receptors with high affinity and show reasonable selectivity for the different receptor subtypes, thus providing reasonable structural leads for the synthesis of new sigma receptor ligands.

In the case of steroid receptors, it has proved useful to assay receptor levels in tumors, because their concentration can provide a useful indicator of the prospects for a favorable response to hormone therapy. Tumor receptor assays were initially done using radioligand binding assays on tumor biopsy samples, but immunoassays are now more commonly used. These assays are not foolproof, however, because the distribution of receptor-positive cells in tumors can be heterogeneous, and thus might be missed by biopsy sampling, and not all tumor sites can even be accessed for biopsy. Also, immunoassays measure a receptor epitope, not a receptor function.

Detecting receptors by imaging with suitable receptor-binding radiopharmaceuticals can provide distinct advantages in assessing the receptors in tumors. Though not as sensitive as ligand binding and immunoassays on tumor biopsy samples, receptor imaging is non-invasive, comprehensive, and physiological; it provides a direct assessment not only of receptor distribution at all tumor sites, but it also assesses receptor "function", at least in the sense of the receptor's activity for ligand binding (Katzenellenbogen et al, 1995). Tumor receptor imaging can be used to identify tumors that are receptor positive, but more importantly, by assessing the level of receptors in tumors, it can provide important information for selecting among alternative antitumor therapies and in monitoring tumor response to therapy.

This chapter will provide a review of the development of radiopharmaceuticals for imaging various receptors that are found in tumors (See Table 1), with a focus on receptors that bind small organic ligands. Receptors that bind peptide hormones, such as somatostatin receptors, are also important targets for tumor imaging, and they are reviewed in another chapter. Important distinctions will also be made between tumor receptor targets that are intracellular, such as the nuclear hormone receptors for steroids and related ligands, and tumor receptor targets that are in the membrane, such as the sigma receptor. The central focus of this chapter will be on the basic characteristics that are required of a radiopharmaceutical for it to be effective in receptor imaging, and the chemical methodology that has been developed to prepare such receptor imaging agents. Actual medical applications of tumor receptor imaging, which in many cases are only beginning to be developed, will also be covered briefly. The author's opinions concerning the directions that the development of receptor-binding radiopharmaceuticals are likely to be taken in the future will be presented as a conclusion.

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE) Table 1. Tumor Receptors that are the Subject of this Review Receptor Type

Receptor

Ligand

Cancer

Nuclear

Estrogen

Estrogens

Breast, Ovarian

Androgen

Androgens

Prostate

Progesterone

Progestins

Breast

Membrane

Glucocorticoid

Corticosteroids

Lymphoma

PPARy

Various

Various

Sigma

Various Amines

Breast and Other

ESSENTIAL CHARACTERISTICS OF THE RECEPTOR-MEDIATED UPTAKE PROCESS AND THEIR IMPLICATIONS IN THE DESIGN OF RECEPTOR-BASED IMAGING AGENTS GETTING SUFFICIENT UPTAKE TO IMAGE: Low RECEPTOR DENSITY AND TARGET UPTAKE LIMITATIONS MEANS THAT VERY HIGH SPECIFIC ACTIVITY is NEEDED Tissue concentrations of receptor are typically very low, in the 1-10 nM range; therefore, receptor-binding imaging agents need to be prepared at very high specific activity in order for a detectable level of activity to be accumulated by a tumor (Katzenellenbogen, 1996; Katzenellenbogen et al., 1982). To detect a 1-cc tumor with a typical tomographic imaging device, it is generally considered that specific activities in the range of 1000 Ci/mmol are required (Katzenellenbogen et al., 1982; Katzenellenbogen, 1992). This is clearly well within the range of the theoretical specific activity of most short-lived radionuclides, and is limited only by the production of the isotope and the development of chemistry for working at the no-carrier-added level. While many effective radiosynthetic methods have been developed, certain types of radiolabeling chemistry, such as electrophilic fluorination (Kilbourn, 1990), do not work well at the tracer level (although progress is being made with this particular radiolabeling method) (Bergman & Solin, 1997). Imaging smaller tumors, as is the case in microPET imaging of rodents, would require considerably higher specific activities (Chatziioannou et al., 1999).

Another limitation to tumor receptor-mediated uptake is the level of fractional saturation that can be achieved in vivo, which is related to the percent of the injected dose that is taken up by the tumor receptor system. Thus, the requirement for high specific activity is linked to whatever limitations there might be to the amount of the injected dose, which are generally based on radiotoxicity considerations, as well as to pharmacokinetic factors. The latter include not only the time available for radiopharmaceutical uptake (i.e., blood-activity curves), but also potential cell membrane barriers to the uptake of radiopharmaceuticals targeted at intracellular receptors (i.e., molecular weight and polarity-lipophilicity relationships; flow-limited uptake). This is less of a consideration with ligands for membrane receptor targets, because those in the periphery can be accessed from the blood pool without traversing a membrane barrier.

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IMAGING TUMOR UPTAKE OVER THE BACKGROUND: ACHIEVING GOOD TARGET TO BACKGROUND CONTRAST REQUIRES HIGH BINDING AFFINITY AND LOW NON-SPECIFIC BINDING Because the factors noted above limit the level of activity that is taken up by tumors through a receptormediated process, it is important that the radiopharmaceutical be designed such that background activity levels will be as low as possible.

One set of factors important to achieving high target-to-background contrast concerns the binding characteristics of the radioligand: These should provide high binding affinity for the receptor and low nonspecific binding (Katzenellenbogen et al., 1982; Katzenellenbogen, 1992). Maintaining high receptor binding affinity with radiolabeled ligands requires careful consideration of ligand structure, including an appreciation of the degree to which radiolabeling by non-isotopic substitution might affect receptor binding. Non-specific binding is usually directly related to lipophilicity; so, achieving low non-specific binding generally means keeping lipophilicity moderate (Katzenellenbogen et al., 1982), although the relationship between trans-membrane uptake into cells and lipophilicity cannot be overlooked (see earlier).

Pharmacodynamic factors can also play a major role in achieving high target-to-background contrast, because when the rate at which intracellular radioligand is bound by the receptor exceeds the rate of its loss from the cell, then "receptor trapping" can occur. This receptor trapping can result in large increases in target-to-background contrast (Frost, 1982).

Finally, pharmacokinetic factors relating to metabolism and clearance have an impact on target to background contrast. Ideally, the radioligand should be cleared at a rate that is sufficiently slow to permit adequate target tissue uptake, but sufficiently fast such that activity in non-target tissues that are close to target sites is cleared within a reasonable time. Also, the processes and routes of metabolism of the agent should not generate radiolabeled metabolites that recirculate and contribute to background activity levels in the region to be imaged.

RECEPTOR STRUCTURE AND IMPLICATIONS FOR RADIOPHARMACEUTICAL DESIGN AND RADIOCHEMISTRY USED FOR THE SYNTHESIS OF RECEPTOR IMAGING AGENTS

NUCLEAR HORMONE RECEPTOR STRUCTURE AND RADIOPHARMACEUTICALS DESIGN It has long been appreciated that the receptors for steroid and related ligands generally have high binding specificity, such that small changes in ligand structure can result in a precipitous loss of affinity (Anstead et al., 1997; Gao et al., 1999). On the other hand, this is not always the case: Sometimes ligands with large substituents or even ligands only distantly related in structure can have high affinity. Therefore, a more accurate term to describe the binding characteristics of these receptors is "selective and eclectic" (Anstead et al., 1997; Gao et al, 1999).

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

719

The cloning and sequencing of nuclear hormone receptors and the more recent X-ray crystal structures of their ligand binding domains now provides a rich bio-informational and protein structural basis for understanding ligand binding characteristics, and for proposing, at least to some degree, ligand design (Weatherman et al. 1999; Wurtz et al., 1996). The ligand is bound within the lower half of a large protein domain of some 250 amino acids that has a canonical "antiparallel a-helical triple sandwich" topology. In most of the structures, the ligand is almost completely surrounded by protein; hence, the large ligand-protein interface can account for some aspects of hormonal specificity.

On the other hand, in many of the structures there are empty pockets that surround the ligand, where there are no protein-ligand contacts (Brzozowski et al., 1997). These regions of the receptor correspond to sites on the ligand where large substituents are often well tolerated, and can even increase binding affinity (Anstead et al., 1997). Also, the lower half of this domain in which the ligand is bound appears to be rather mobile, and residue positions, even the position of helix backbones, can shift to a considerable degree to accommodate ligands with large substituents or ligands having different shape and sizes (Brzozowski et al., 1997; Shiau et al., 1998). In some cases, substituents on ligands can be sufficiently long so as to emerge from the protein interior and gain access to the solvent (Brzozowski et al., 1997; Pike et al., 1999; Shiau et al., 1998).

These unexpected opportunities for ligand binding, which, truthfully, have been discovered more by serendipity than by design, have been termed "preformed pockets" and "deformable pockets", and are thought to account for the eclectic nature of the specificity of these nuclear receptors (Anstead et al., 1997). They do, however, provide suggestions for sites on the ligand at which the addition of substituents might improve the binding or pharmacokinetic characteristics, or where bulky radionuclide or radiolabeling units might be accommodated without markedly reducing binding affinity (Katzenellenbogen et al., 1982).

Sequence comparisons among nuclear hormone receptors has allowed them to be classified into evolutionally related families (Laudet, 1997). Because similarities in sequence often translate into similarities in function and possibly binding preferences, sequence similarities can alert one to situations where ligand binding might occur in a crossed or heterologous manner. An example of this is the similarity in ligand binding selectivity among members of the glucocorticoid nuclear receptor family that includes receptors for progestins, androgens, and mineralocorticoids. When it is not recognized, such crossed binding specificity can considerably confound the interpretation of images of specific receptors.

MEMBRANE RECEPTOR STRUCTURE AND RADIOPHARMACEUTICAL DESIGN Structural information on membrane receptors is much less developed than with nuclear receptors. There are many different protein topologies and motifs that are associated with membrane receptors: Those that respond to large peptide or protein hormones, such as the growth-factor tyrosine-kinase or the cytokine nontyrosine kinase receptor classes; those that respond to small molecules, such as neurotransmitters, and those that are ligand-gated ion channels or other transporters.

720

HANDBOOK OF RADIOPHARMACEUTICALS

Based on what is known about their sequences and sizes, the three sigma receptors, which will be discussed briefly in this chapter, appear to be members of different structural classes. Nothing is known about their structure, although they have been speculated to be G-protein coupled receptors or regulators of ion channels (Seth et al., 1998; Vilner & Bowen 2000). If their structural classes could be determined with greater certainty, then it might be possible to generate homology models that could be useful in assessing the structures of potential ligands.

Two APPROACHES TO THE DESIGN OF RECEPTOR-BINDING RADIOPHARMACEUTICALS: "PENDANT/CONJUGATE DESIGN" vs THE "INTEGRATED DESIGN"

THE

It is useful to make a conceptual distinction between two limiting concepts for receptor-binding radiopharmaceutical design, based on the relationship between the portion of the ligand that is responsible for binding to the receptor and the portion that embodies the radionuclide (Hom & Katzenellenbogen 1997b). These are illustrated schematically in Figure 1. In the "pendant/conjugate design", the receptor-binding (i.e., ligand) unit and the radiolabeled unit are in separate, discrete structural and functional moieties that are simply attached together. This is the design

Figure 1. Two concepts that guide the design of receptor-binding radiopharmaceuticals. that is typically used when the radiolabeled unit is large, such as a complex between a radiometal and a multidentate chelate. This approach provides flexibility, because each unit can be designed separately and optimized for its own function: The ligand can be designed to achieve high affinity for the receptor, and the radiolabeled unit to accommodate the radionuclide in an optimal fashion. However, when the radiolabeled unit is quite large, both it and the tether, through which it is attached to the binding unit, can potentially interfere with the ligand unit binding to the receptor. Since radiopharmaceuticals of the conjugate design are typically quite large overall, this can lead to undesirable pharmacokinetic behavior and limit membrane transport.

In the "integrated design", as indicated by the name, the functions of the ligand binding and radiolabeled units are integrated within one structure. The ultimate goal in the integrated design is a receptor ligand isotopically labeled (i.e., with a radioisotope of one of the normal constituent elements of the ligand, such as carbon-11) or non-isotopically labeled with small, single-atom radionuclides such as fluorine-18. Larger radiolabeled units can be incorporated; the only requirement is that the radionuclide and the constituent atoms associated with its attachment to the ligand be in a part of the molecule that is integrally involved in its binding to the receptor. As will be shown, both inorganic and organometallic metal complexes, even ones that are quite large, can be incorporated within the receptor-binding unit of the radiopharmaceuticals.

'21

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE) RADIOCHEMISTRY USED FOR THE SYNTHESIS OF RECEPTOR IMAGING AGENTS

LABELING RECEPTOR-BINDING RADIOPHARMACEUTICALS WITH RADIOHALOGENS Because monoatomic labeling can be achieved with halogen radioisotopes, most of the halogen-labeled radiopharmaceuticals can be considered to be integrated radiopharmaceuticals.

The halogen substituent,

whether a single atom or a haloalkyl or halovinyl group, typically is close to the core of the ligand and makes a significant contribution to the binding of the radiopharmaceutical to the receptor.

BROMINE AND IODINE RADIOISOTOPES Radioisotopes of bromine and iodine provide a nice combination of convenient half-life, high specific activity, and versatile chemistry. They have been used extensively for the labeling of steroidal and nonsteroidal ligands for nuclear hormone receptors, and ligands for the sigma receptor.

Nucleophilic

displacements with bromine and iodide ions can be used to introduce the halogen at aliphatic positions, and electrophilic halogenation, typically in halodestannylation reactions, can be used to label at vinylic and aromatic positions. One limitation of radiolabeling with bromine and iodine is that these halogens are relatively large. Therefore, careful consideration needs to be given to the sites in the receptor ligand where these halogens can be substituted to avoid interference with receptor binding. Clinical studies have been done with some of these agents and are discussed briefly later in this chapter. Ligands for the estrogen receptor have been labeled with these radiohalogens at the 16a position, and on vinyl substituents at the 17ct position. Some examples of these radiopharmaceuticals, and their routes of synthesis, are shown in Figure 2. More extensive reviews are available (Cummins, 1993; Katzenellenbogen, 1996).

2- LiAIH

4

(|

H O

16 a -Bromoestradiols = -H -OMe -Et

R =H R = OMe

140/8.4/16 20/12/26

lodide/oxictant

H Me 3Sn Me 3Sn H

H I

HO

1

H

33/1.6/3.1 47 / 4.0 / 9.5

17 a -lodovinylestradiois |RBA/%ID»g

•' /(targefcmuscle)

|

Figure 2. Structures of representative radiobromine- and radioiodine-substituted steroidal estrogens and their routes of synthesis by nucleophilic iodide displacement or electrophilic halogenation of alkenes or vinyl stannanes. In this figure and following figures, some information of the receptor binding and tissue distribution is provided in the following summary form: RBA / %IDg-1 / (target:muscle). The RBA is the relative binding affinity, where the parent ligand, in this case estradiol, has an affinity of 100. The %ID»gm-1 is the uptake at 1 hr in the principal target tissue, in this case the uterus, in rats of ca. 50 g weight. The target:muscle ratio (a measure of contrast) is the ratio of activity taken up at 1 hr in these tissues.

722

HANDBOOK OF RADIOPHARMACEUTICALS

The 16ct position in estrogens is very tolerant of these large halogens; in fact, the bromine and iodine analogs actually have equivalent or even greater affinity than do the parent ligands. By contrast, the epimeric 163 bromo and iodoestrogens (not shown) have markedly reduced affinity. In some cases, additional substituents have been introduced into the 11P position (or other positions) of these bromo and iodo estrogens to increase their binding affinity and/or reduce their lipophilicity, and these changes have generally increased their target tissue uptake (Ali et al., 1993a, 1993b; Katzenellenbogen, 1996; McElvany et al, 1982).

Radiolabeling at the 16a position can be done either by nucleophilic displacement of a 163 leaving group with iodide ion to give the 16ct-iodoestrogen or by electrophilic bromination of an estrone enol derivative, which gives the 16a-bromoestrone, followed by reduction of the 17-ketone to give predominantly the 17p alcohol (Hochberg, 1979; Katzenellenbogen et al., 1981). Both methods work well and give products that can be readily purified to high radiochemical and chemical purity. The electrophilic approach to prepare the 16a-iodoestrogen works well though the halogenation step, but deiodination occurs during the ketone reduction. The 16a-bromoestrogens have not been prepared by the nucleophilic route, although there is no apparent reason why this approach would not work.

The 17a-iodovinyl estrogens can be readily prepared by electrophilic halogenation of a vinyl stannane or vinyl boronic acid precursor (Ali et al., 1988; Hanson & Franke, 1984; Hanson et al., 1988, 1989, 1990, 1998; Hughes et al., 1993; Jagoda et al., 1985). In the former case, both E- and Z-isomers have been explored. The Z-isomer has higher affinity for the estrogen receptor, but is less stable (Hughes et al., 1997; Rijks et al., 1996). In vivo, a surprising Z to E isomerization occurs, so that regardless of which isomer is administered, it is the E isomer that accumulates in target sites (Hughes et al., 1997).

Iodine substitution on the aromatic ring of the non-steroidal estrogen hexestrol led to one of the first estrogen radiopharmaceuticals, o-iodohexestrol (Figure 3) (Katzenellenbogen & Hsiung, 1975). The affinity of this ligand was quite good, but its lipophilicity and binding to serum proteins was too high for it to be effective in imaging (Katzenellenbogen et al., 1975). Iodine and bromine are better accommodated on the aliphatic side chains of hexestrol and hexestrol analogs, and on certain other non-steroidal estrogens (Desombre et al., 1995;Landvatter et al., 1982).

= -Br,-CHzBr X= -l.-CH 2 I X = I 130/16/9.8

Figure 3. Iodine and bromine-substituted non-steroidal estrogens. For a definition of RBA / %IDg- l / (target:muscle), see the legend to Scheme 2. In this case, the parent compound is estradiol, the target tissue is the uterus, and 50 g immature female rats were used.

Iodine substitution has also been used to label androgens and progestins. The 16 position proved to be less favorable for iodine substitution in androgens (Hoyte et al. 1982, 1985), but the 7a and 17a positions are quite tolerant (Labaree et al., 1997, 1999). The structures of iodoandrogens are shown in Figure 4. These

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

723 OH

16a-I RBA=1 X = 16 ct/P -Br RBA = 3

O**^*f^S"*-CH3

RBA = 11

OH »R

RBA = 54 = CH3 71/0.39/2.5

1 JL I O*^-**^-"^ "'I

R= H RBA = 47 R= CH3 RBA=25

Figure 4. Iodine and bromine-substituted androgens. In most cases only RBA values are given, relative to R1881. For a definition of RBA / %IDg-11 target:muscle, given in one case, see the legend to Figure 2. In this case, the target tissue is the ventral prostate, and 200 g diethylstilbestrol-treated mature male rats were used.

compounds are prepared by nucleophilic iodide ion displacement of a suitable sulfonate ester or by electrophilic iododestannylation, methods similar to those used to prepare the bromo and iodoestrogens (above). The 7a-iodo compounds are reported to be somewhat unstable in vivo, and, as a consequence, the use of the 7a-fluoro analogs (Figure 10) has been recommended (Garg et al., 2001; Labaree et al,, 1997, 1999).

A number of iodine-substituted progestins have been described. These are shown in Figure 5 (Rijks et al., 1998; Van Den Bos et al, 1998). Bromine and iodine substitution of other nuclear hormone receptor ligands is much less developed. An iodine-substituted ligand for the peroxisome proliferator-activated receptor gamma (PPAR7) is known, but its affinity for this receptor is only modest (Young et al., 1998).

XaH.H 66/-/138/0.38/3. X a =CH, 137 / 0.34 / 2.6167 / 0.71 / 7

Figure 5. Iodine-substituted progestins. For a definition of RBA / %lDg- l I (target:muscle), see the legend to Figure 2. In this case, the parent compound is R5020, the target tissue is the uterus, and estrogen primed female rats were used; uptake was at 2 hours.

Sigma receptors bind a large variety of amines, and different receptor subtypes are distinguished by differences in their relative affinity for certain ligands, the sigma-1 receptors binding preferentially (+)pentazocine (Choi et al., 2001) and sigma-2 receptors binding preferentially di-o-tolylguanidine (Wilson et al., 1991). Higher affinity ligands for both receptors have been reported (Figure 6, and later, Figure 13, and

HANDBOOK OF RADIOPHARMACEUTICALS

724

O K1= 11nM( ol) (John et al. 1999 a orb)

210nM(

(+)pentazocine (Choi et al.. 2001)

1900 nM(

o2)

(John et al 1996)

ol) o2)

K1=0.38llM(

21 nM(

o1)

o2)

2-<Waterhouse et al. t997a.b.c)

(Gamer et al,.. 1994)

(Walorhouseelal..

,997 a,b)

Sigma-2 Racaptor Ligands

CH3

Di-o-tolyl-guanidine

Kd = 38 nM( (Wilson et al.. 1991)

18 nM(

o1) o2)

2-1 K1 = 6.2 nM( o2) 3-1 K1 = 8.0 nM ( o 2 ) 4-1 K1 = 2.8 nM (o2) (Wilson et al. 1991)

Figure 6. Representative iodine and bromine-substituted ligands for the sigma receptors. Also shown are the structures and affinities of the parent ligands used to define sigma-1 (01) and sigma-2 (02) binding sites, (+)-pentazocine and di-otolyl-guanidine, respectively. Where reported, affinities of the halogen-substituted ligands are given (as Ki or Kd values).

references noted in association with these figures). Because of the generally wide binding specificity of these receptors, iodine has been used extensively to radiolabel sigma receptor ligands, although bromine substitution is also well tolerated. Examples of both types of halogen-substituted ligands are shown in Figure 6 (Gamer et al., 1994; John et al., 1999a, 1999b; Waterhouse et al., 1997a, 1997b, 1997c) and they are prepared by electrophilic halodestannylation reactions.

There is little doubt that labeling receptor ligands with bromine and iodine radioisotopes can be both convenient and effective. Given the newly expanding availability of some other radioisotopes of these elements, including positron emitting ones such as bromine-76 and iodine-124, coupled with the new opportunities for imaging in small animals, it is likely that the use of these radionuclides will see a sharp increase in the future.

FLUORINE-18 LABELING Fluorine-18 has proved to be one of the most robust and versatile radionuclides for the labeling of receptor ligands, and it has seen very widespread use. Its small size makes fluorine an isostere of a hydrogen or a hydroxyl group, so fluorine can often be substituted at many different sites in a ligand with only minimal effect on receptor binding affinity and pharmacokinetics. Also, the great stability of the carbon-fluorine bond is an asset during synthesis and in vivo disposition.

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

725

Fluorine-18 is readily available as fluoride ion (and reagents derived therefrom) in high specific activity, and its half-life is sufficiently long to permit complex synthesis and careful purification; imaging can also be done after considerable post-injection delays that may be required to achieve adequate distribution or target to background contrast development.

The only limitation of significance in radiofluorination is that

electrophilic fluorinating agents, typically derived from fluorine gas, are not available at sufficiently high specific activities required for receptor-based imaging, although this situation is improving with the development of microscale post-bombardment methods for fluoride ion/fluorine gas exchange (Bergman & Solin, 1997; Kilbourn, 1990). Examples of radiolabeled estrogens, androgens, progestins, corticosteroids, and ligands for other nuclear receptors, and the syntheses used to prepare some of these agents, are shown in several Figures that follow (Figure 7-9).

A brief review of clinical uses of fluorine-substituted receptor

ligands is presented later in the chapter.

= H,Me R3=H,C=C-H

Figure 7. Fluorine-substituted estrogens. Information on the estrogen receptor binding affinities and target tissue uptake characteristics can be found in the references cited, or in a recent review article (Katzenellenbogen, 1996).

Fluorine labeling of estrogens has been studied most extensively, and there are many examples of estrogens having this halogen at the 2, 16a and 16B positions, and on 7a-alkyl and 11B-alkyl and alkoxy substituents, that retain high affinity for the estrogen receptor (Figures 7 and 8) (French et al. 1993a, 1993b; Hostet'er et al., 1999; Kiesewetter et al., 1984a, 1984b; Pomper et al., 1990; Vanbrocklin et al., 1990; 1992, 1993a, 1993b, 1994). Undoubtedly, substitution at other sites would probably also be well tolerated. Fluorine ion substitution of reactive sulfonate esters, or in some cases cyclic sulfates, was used to prepare all of the fluoroestrogens (Figure 8), except the C-2 substituted analog (Figure 9) (Kilbourn, 1990). Typically, with the more reactive sulfonates, conditions could be found so that the displacement reactions took place rapidly.

Good examples of these reactions are presented in Figure 8, in which the preparation of the two most important fluorine-18 labeled estrogens, 16a-[I8F]fluoroestradiol (FES)(Kiesewetter et al., 1984b) and 16$[18F]fluoromoxestrol (BFMOX) (Vanbrocklin et al. 1993b), is shown. The highly reactive 16j3-trifloxyestrone 3-triflate undergoes substitution with tetrabutylammonium [18F]fluorine ion to give the 16afluoroestrone product within seconds at room temperature. Subsequent hydride reduction generates the required 17p alcohol and deprotects the phenol, giving the very useful radiopharmaceutical agent FES for short (Kiesewetter et al., 1984b). The whole synthesis, from preparation of the anhydrous organic soluble fluoride ion through the final HPLC purification, can be completed within less than one half-life, using a simple automated synthesis and purification system (Brodack et al., 1986). The preparation of PFMOX proceeds in a similar fashion (VanBrocklin et al., 1993b).

726

HANDBOOK OF RADIOPHARMACEUTICALS

16a-{18F)Fluofoestradiol (FES)

CHjO

16^-{18F]Fluoromoxestrol @FMOX)

10 /18 / 24

Figure 8. Structure and synthesis of two important fluorine-18 labeled estrogens, FES and PFMOX. For a definition of RBA / %ID.g.1 / (targetrmuscle), see the legend to Figure 2. In this case, the parent compound is estradiol, the target tissue is the uterus, and 50 g immature female rats were used.

Radiolabeling at an aromatic position with fluorine-18 at high specific activity presents a special challenge. One might think that electrophilic fluorodestannylation or desilylation methods would be appropriate, but currently these methods cannot be used to prepare radiopharmaceuticals at sufficiently high specific activity for receptor imaging on a routine basis (Bergman & Solin, 1997; Kilbourn, 1990). The incorporation of fluoride ion into aromatic systems by the Balz-Schiemann or related reactions, which involves the decomposition of aromatic diazonium fluoride derivatives, works very well on the macroscopic scale, but yields drop precipitously when the reaction is conducted on the tracer level (Haroutounian et al., 1991; Kilbourn, 1990). What has proved most effective for aromatic radiofluorination is nucleophilic aromatic substitution with fluoride ion. Effective methods for nucleophilic aromatic radiofluorination have been worked out in a number of systems, and the minimal requirement is known to be a good leaving group (such as nitro, trimethylammonium or dimethylsulfonium) and an electron-withdrawing activating group (such as a carbonyl and nitrile function) situated at a position ortho or para to the leaving group (Hosteller et al., 1999; Kilbourn, 1990).

An example of this approach that was developed in our laboratory for the synthesis of 2-[l8F]fluoroestradiol is shown in Figure 9. It involves the synthesis of the 6-keto-2-trimethylammonium analog of estradiol, in which the 6-keto group provided an activating group para to the leaving group, as the substrate for the nucleophilic aromatic substitution reaction. Reaction with [18F]fluoride ion then took place readily at the C-2 position, and the activating and protecting functions were removed efficiently and quickly in two subsequent steps (Hosteller et al. 1999).

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE) OCH3

727 OCH3

2. AIBr3, EtSH

HO' 18

2-[ F]Fluoroestradiol (2FES) RBA = 87

Figure 9. Synthesis of 2-[18F]fluoroestradiol (2FES), an example of nucleophilic aromatic substitution. For a definition of RBA, see the legend to Figure 2. In this case, the parent compound is estradiol.

A number of sites for fluorine substitution on androgens have been explored, and examples of fluoroandrogens are shown in Figures 10 and 11. The best position for fluorine substitution appears to be at 7a, 16ß and possibly 11ß. As were the estrogens, most of the fluoroandrogens were prepared by fluoride ion displacement reactions on a reactive sulfonate esters or cyclic sulfates. Two examples of these syntheses are shown in Figure 10 (Garg et al., 2001; Labaree et al., 1997, 1999; Liu et al., 1991, 1992a, 1992b).

Synthesis of the 6a-fluoro and 1 ip-fluoro androgens, however, required a different approach that involved a halofluorination reaction (Choe et al., 1995; Choe & Katzenellenbogen, 1995). As illustrated in Figure 11 for 1 1ß-fluoro-Sa-dihydrotestosterone, a precursor with a 9(11) unsaturation, underwent a bromofluorination reaction to give the 11 ß-fluoro-9a-bromo adduct. The bromofluorination reagent is generated in situ from anhydrous fluorine ion and an electrophilic brominating agent such as dibromodimethylhydantoin, under acidic conditions (Brandes & Katzenellenbogen, 1987; Chi et al., 1986, 1995; Choe et al., 1995). This formal anti-Markownikow addition involves bromine as the electrophile and fluorine as the nucleophile.

The halofluorination reaction works well in model systems, and on the tracer scale is a much more favorable process than is hydrofluorination (Chi et al., 1986, 1995). The reason for this is that in bromofluorination the alkene nucleophile, a soft base, is better matched with the bromine electrophile, a soft acid, than it is with a proton, which is a hard acid. In fact, the use of a proton as the electrophile is highly counterproductive in tracer level hydrofluorination, because it is a hard acid and preferentially interactions with fluoride, a hard base, deactivating it, rather than interacting with the soft base alkene. The 9a-bromo substituent present in the initial bromofluorination product is readily removed by radical tin hydride reduction, giving the desired 11ß-fluoro androgen.

Nearly all of the fluorine-substituted progestins (Buckman et al., 1994, 1995; Kochanny et al., 1993), corticosteroids (Pomper et al., 1992), and ligands for the PPARy receptor (Kim et al., submitted), shown in Figure 12, were prepared by fluorine ion-sulfonate ester displacement reactions. Further details about the synthesis and biodistribution of these compounds can be found in the references cited.

Quite a number of fluorine-substituted ligands for the sigma receptors have been prepared (Dence et al., 1997; Shiue et al., 1997; Waterhouse & Collier, 1997). Several of these are shown in Figure 13, and they

728

HANDBOOK OF RADIOPHARMACEUTICALS

= H; RBAz 78 = Me; RBA = 123 RBA (DHT) s 100

,.OTt 2. UAIH 4 3. HO

FDHT 43/0.43/7.6

H ,-CH,1§F

CH,

20-FMb 53/0.97/3.8

Figure 10. Structure and synthesis of fluoroandrogens. For a definition of RBA / %ID»g*1 / (target:musck), see the legend to Figure 2. In this case, the parent compound is R1881 (unless indicated to be Sa-dihydrotestosterone, DHT), the target tissue is the ventral prostate, and 200 g mature male rats pretreated with diethylstilbestrol were used. Binding and tissue uptake data on other compounds can be found in the references cited.

were prepared by fluoride ion displacement reactions on reactive sulfonate precursors or by nucleophilic aromatic substitution. In the case of the sigma-1 selective ortho and para-fluorobenzyl analogs, these compounds were prepared in fluorine-18 labeled form by nucleophilic aromatic substitution on the

53IOA9I&2

Figure 11. Synthesis of a 11ß-fluoroandrogen by a bromofluorination-debromination sequence. For a definition of RBA / %ED«g"1 / (targetrmuscle), see the legend to Figure 2. In this case, the parent compound is R1881, the target tissue is the ventral prostate, and 200 g mature male rats pretreated with diethylstilbestrol were used.

benzaldehyde precusors, followed by a one-step reduction-iodination with diethyldiiodosilane, to give ortho or para-fluorobenzyliodides, which were then reacted with the secondary amine precursor to give the desired compounds (top half of figure) (Dence et al., 1997). A related nucleophiiic aromatic substitution route was also used to prepare the sigma-2 guanidine analogs (bottom half of figure) (Wilson et al., 1991). Remarkably, some of the highest affinity and most sigma-2 selective ligands have not been prepared in fluorine-18 labeled form (Moltzen et al., 1995; Perregaard et al., 1995; Sanchez et al., 1997).

729

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE) Fluoroprogestlnsj

R = H (FFNPAcetal)190/6.8/18 R = Me (FFNP Ketal) 173/6/5/20

X = OH ORG2Q5«204/3,4/15) X = F FENP J897/4.6/41)

[RBA / %ID-gni' / (uterus:muscle)]

X

RBA

X

RBA

H

146 (RU 36752)

H

192 (RU 28362)

F

65

F

59

H

F

1030 (Trlamcin. Actn.)

170

| Fluorine-Substituted PPAR Ligands | CO2H -CHjCHjF -O-Ph -CH» -OCHtCH/

17 nM 7 nM

Figure 12. Structures of fluoroprogestins, fluorocorticosteroids, and fluorine-substituted ligands for the PPARy receptor. For a definition of RBA / %JD*gl I (target:muscle), see the legend to Figure 2. In the case of the progestins, the parent compound is R5020, the target tissue is the uterus, and 50g female rats pretreated with estradiol were used.

ISOTOPIC LABELING OF RECEPTOR-BINDING RADIOPHARMACEUTICALS WITH CARBON-H Isotopic

labeling of endogenous receptor

ligands with carbon-11

to prepare

receptor

binding

radiopharmaceuticals should have no consequences in terms of the binding affinity and pharmacokinetic behavior of the ligand. The challenge here is all in the synthesis; typically, introduction of a carbon atom within the core of a polycyclic system is a major undertaking, especially considering the short half-life of carbon-11. Thus, most carbon-11 labeled radiopharmaceuticals are synthetic ligands, in which the labeled atom is positioned at a site where it can be incorporated readily. Radiolabeling with carbon-11 is very common in neuroreceptor ligands where heteroatom methyl (i.e., N-, O-, and S-methyl or other small alkyl) groups are frequently found.

These ligands can be labeled

with carbon-11 by alkylation of the corresponding nor-compounds with [11C]methyl iodide. By contrast, C} 1 labeling of nuclear hormone and sigma receptor ligands is far less developed.

HANDBOOK OF RADIOPHARMACEUTICALS

730

(Denceetal., 1997)

•CHO

,CHO

2.2.2 Kryptofix .^^^> X,Y = -H,-NOior-NO2,-H

.CHjl Di«(Jos«ane

X.Y = -H,.For-F,-H

X.Y = -H.-F Of -F,-H

2-F 3-F 4-F 2-M4.4-F

K|= 8nM|o2) K,= 13nM(o2) K,= 9nM(o2) K,= 3.2nM(o2)

(Wilson et at.. 1991)

OGL 4 17 nM 3 10 nM 2 15 nM 1 3 nM

0.12 nM

2.8 nM 3.0 nM 4.7 nM

(Pgrrggaard af a/.. (995; MoKzon et al.. 1995: Sanchez el al., 1997)

Figure 13. Fluorine-substituted ligands for the sigma receptors, and nucleophilic aromatic substitution routes used to prepare 4-['8F]fluorobenzyl (ol, middle) and[l8FJfluoroaniline (o2, bottom) units.

As shown in Figure 14, a 17a-[!1C]methyl group has been introduced into steroids by the addition to the 17 keto group of ["CJmethyllithium, which is generated quite easily from methyl iodide by exchange with butyllithium (Dence et al., 1996). In the estrogen series, this approach gave some high affinity ligands (Napolitano et al. 1995a; 1995b). An unusual electrophilic methoxylation reaction, in which methoxyfluoride, generated from methanol and fluorine gas, can be used to introduce a [11C]methoxy group (not shown) (Jonson et al., 1998). This reaction has been used in the estrogen series, but the products had low binding affinity (Jonson et al., 1998).

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

731

1. 11CH3I, BuLi 2. HCI

R = H 57 / 4.5 / 5.7 R = Et 83/6.6/13.4

R = H, Et

Figure 14. Structures of carbon-11 labeled estrogens. For a definition of RBA / %ID.g.1 / (target:muscle), see the legend to Figure 2. In this case, the parent compound is estradiol, the target tissue is the uterus, and 50 g immature female rats were used.

The synthesis of a few C-11 labeled sigma receptor ligands has been described (Figure 15). One of the sigma-1 selective ligands was labeled at two sites, using either [11C]methyl iodide or l-[11C]propyl iodide (Ishiwata et al., 1998; Kawamura et al., 1999, 2000).

Despite the limited number of examples of C-11 labeled radiopharmaceuticals directed at tumor receptor targets prepared to date, this approach merits further investigation. In many cases, it should be possible to prepare high affinity analogs of receptor ligands having MeO- or MeS- substituents that could be readily prepared by reaction of the nor compounds with [11C]methyl iodide. The only proviso to keep in mind in the development of C-l1 labeled tumor receptor ligands is that because of the short half-life of the

X,Y = C-11, orC-12,C-11

(Kawamura et al.. 1999,2000)

Act-NH-CN Sj!^ OH

TBA-OH

Xj*** O11CH3

(Wilson etal, 1991)

H311CO' 2-OMe K, = 17nM(o2) 3-OMe K,= 7nM(o2) 4-OMe K, = 10nM(o2)

Figure 15. Carbon-11 labeled ligands for the sigma receptors.

radionuclide, the pharmacokinetic features of the radiopharmaceutical have to be such that they are cleared rapidly from non-target areas in the region to be imaged.

LABELING RECEPTOR-BINDING RADIOPHARMACEUTICALS WITH RADIOMETALS

TECHNETIUM RADIONUCLIDES The ready availability of technetium-99m from the molybdenum-99/technetium-99m generator system places a premium on the development of any good radiopharmaceutical that can be labeled with this radionuclide.

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HANDBOOK OF RADIOPHARMACEUTICALS

Improvements in production methods for the positron-emitting technetium radioisotope, Tc-94m, has further increased interest in technetium radiochemistry (Qaim, 2000).

Being a metal, technetium requires multi-atom units to be stably affixed to an organic molecule. This can be done either through a sizable inorganic multidentate heteroatom chelate system, such as the N2S2 chelates, or through a multi-atom organometallic binding unit such as a cyclopentadienyl an ion or other sets of small donor molecules. The size of the metal binding unit suggests the favorability of using the pendant/conjugate approach for the design of receptor-binding radiopharmaceuticals, and this has proved to

HBA (R5020) « 100

^^^^

OH

.--> * = °*«1 ^?

rv^

1 JL T

"S'£"3

HO^^^i

x

^siy S**f^f~^

= ^s*t*-o

RBA, 43

*= ss-A«-s

>>-

^ RBA = 11 ^v/^

RBA(E*tradk>Q*100

Orf CO RBA = 14

RBA * 12

R^COfc RBA = 5

RBA * 29

Figure 16. Structures of steroid pendant/conjugate metal (technetium or rhenium) chelate systems in the progestin (top), androgen (middle) and estrogen (bottom) series.

be effective for the design of technetium-labeled ligands for neuroreceptor transport systems (Horn & Katzenellenbogen, 1997b). The development of technetium-labeled radiopharmaceuticals of the integrated design presents a particular challenge, because it is not obvious in what way the sizable metal chelate or organometallic system might mimic the structure of a receptor ligand.

Progestins, androgens, and estrogens labeled with technetium or rhenium inorganic or organometallic units in a pendant/conjugate fashion are shown in Figure 16.

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RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

Because of the known tolerance of the steroid receptors for large substituents at the 7a, 11ß and 17a positions, we prepared two types of pendant radiopharmaceuticals, progestins having a tetradentate oxorhenium complex tethered to the 11ß position (Dizio et al., 1991, 1992; O'Neil et al., 1994), and androgens (Wust et al., 1997) and estrogens having various inorganic and organometallic rhenium units tethered to the 17a or 7a positions, including a cyclopentadienyl rhenium/technetium tricarbonyl system (Cp-Met(CO)3) (Skaddan et al, 1999, 2000; Wust et al., 1996, 1998).

The inorganic complexes were

prepared by the reaction of a suitable rhenium or technetium donor reagent (typically Met(O)Cl4" for the oxometal systems and Met(CO)3* for the metal tricarbonyl systems) with an appropriate heteroatom chelating unit.

The Cp-Met(CO)3 derivatives were prepared by one of several methods that begin with either

peitechnetate or perrhenate, or the prereduced metal tricarbonyl cation precursor, and use a suitable cyclopentadiene donor (Minutolo & Katzenellenbogen, 1999; Spradau & Katzenellenbogen 1998).

Many of these radiopharmaceuticals retained high affinity for their cognate receptors, but those that were prepared in Tc-99m labeled form failed to show receptor-mediated distribution in vivo, presumably because they are large and very lipophilic, and their affinities, though respectable, are not very high. Agents of this design need to be explored further, but their ultimate promise is in question because of their overall large size. [ParentLigand {

| Integrated Analog}

OH

K H 5u-Dihydrotestosterone (DHT) ,OH

Non-Steroidal Estrogen

[3 * 1] HS3 Comple

HO'

Tetradentate N:S2 Complex X =O (unstable) X = H.H (moderately stable)

Figure 17, Structures of integrated inorganic metal (technetium or rhenium) chelate systems (right) that mimic estrogens (left).

We have prepared two types of integrated ligands for the estrogen receptor, inorganic complexes and organometallic systems.

The inorganic rhenium complexes are illustrated in Figure 17. In these, the

complex itself replaces two of the central rings of the steroid ligand. These complexes are prepared by treating the heteroatom chelate system with a suitable metal precursor such as Tc(O)Q4~ or (Ph3P)2Re(O)Cl3. Although they are close structural mimics of the parent estrogens, none of these inorganic complexes were

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HANDBOOK OF RADIOPHARMACEUTICALS

stable under physiological conditions (Horn & Katzenellenbogen, 1997a; Skaddan & Katzenellenbogen, 1999; Sugano & Katzenellenbogen ,1996).

The integrated organometallic systems (Figure 18) have a very different geometry; they are aryl-substituted cyclopentadienyl rhenium tricarbonyl systems, having a "piano stool" organometallic unit that forms the core of the ligand. These first generation ligands do have significant affinity for the receptor, albeit, at this point, still low (Mull et al., submitted). The unusual structure of these systems may make it seem strange that they bind to the estrogen receptor at all. However, the ligand binding domain of this receptor has a remarkable amount of unoccupied space (200A3), and it is known to bind ligands based on adamantane and 1,12decacarborane (Endo et al., 1999).

Efforts to prepare integrated, technetium-labeled ligands for nuclear hormone receptors have, thus far, been very limited, and a clear success in the development of such radiopharmaceuticals has not yet been achieved. Despite the strictures of hormone binding specificity of the nuclear hormone receptors, and probably because of their eclectic nature, it should be possible to prepare high affinity technetium labeled radioligands that will be useful in imaging tumor nuclear hormone receptors. R,

R, RBA

Et pOH-Ph 23% H pOH-Ph 11%

Figure 18. Structures of integrated organometallic (technetium or rhenium) systems that mimic estrogens. RBA is relative binding affinity, where RBA(estradiol) = 100%.

The wide tolerance of the sigma receptors for alterations in ligand structure has permitted the preparation of technetium-labeled compounds that have high affinity for this target. Two of these agents are shown in Figure 19, and it is likely that many more technetium-labeled ligands having high affinity for this receptor could be prepared. CHS

Kd = 49-59 nM (ol + o2)

Kd = 2700 nM (a 22 nM (a 2)

Figure 19. Technetium-99m-labeled ligand for the sigma receptors.

GALLIUM RADIONUCLIDES There are a number of useful gallium radioisotopes, but using these in the development of tumor receptor imaging agents has proved to be a challenge. Stable inorganic complexes of the gallium(III) ion typically involve tetra- to hexadentate chelates that are large and, therefore, unlikely to be suitable for the construction

735

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

of tumor receptor ligands of the integrated design (Jones-Wilson et al., 1995; Sun et al., 1996). Although they have not been investigated so far, these inorganic gallium complexes might be suitable for use as pendant radiolabeling units in radiopharmaceuticals of the conjugate design.

Pendant/Conjugate Design

n = 6 or 10

CH3 Centchromene

CH

Dimethylgallium Analog

Figure 20. Structures of integrated and pendant/conjugate gallium complexes designed to mimic estrogens.

compounds that have been reported are shown in Figure 20. In the integrated agent, the dimethylgallium was to play the role of an isopropylidene unit, of which it is a close stereochemical mimic. However, in both this compound and the conjugate agent, the dimethylgallium cation was tethered to a bidentate salicylaldimine unit that did not form a complex of sufficient kinetic stability to survive physiological conditions.

A BRIEF SUMMARY OF MEDICAL APPLICATIONS OF TUMOR RECEPTOR IMAGING There are a number of comprehensive reviews on the use of steroid receptor imaging agents in cancer detection and management (Jonson & Welch, 1998; Katzenellenbogen et al., 1982, 1992, 1995, 1996; Scheidhauer et al., 1998). Therefore, only a brief overview will be provided in this chapter.

IMAGING ESTROGEN RECEPTORS IN BREAST CANCER Of all the non-peptide receptors in tumors, the estrogen receptor has been the most extensively studied by imaging. The first imaging of a breast tumor through the estrogen receptor was done using 16a[77Br]bromoestradiol (McElvany et al., 1982). Image quality was low, because of the high energy of this yemitter, but subsequent studies using 16a-[I8F]fluoroestradiol (FES) and various iodine-123 labeled estrogens, showed clear images of both primary and metastatic breast tumors (Dehdashti et al., 1995, 1999; Kenady et al., 1993; McGuire et al., 1991; Mintun et al., 1988; Preston et al., 1990; Ribeiro-Barras et al., 1992; Rijks et al., 1997; Scheidhauer et al., 1991; Schober et al., 1990). In an effort to develop an imaging agent superior to FES, many F-18 labeled estrogen analogs were prepared and their tissue distribution studied in rats. The best of these, 16p-[18F]fluoromoxestrol, looked very promising in animal studies; surprisingly, it proved to be unsatisfactory for breast tumor imaging in humans (Jonson et al., 1999; VanBrocklin et al., 1993b). The reason for the failure of this compound in humans is not known, but it may be related to its very low affinity for sex hormone binding globulin, a serum steroid transport protein that is absent in rats but present in humans, where it might be playing a role in the distribution and target uptake of the radiopharmaceutical (Bonasera et al., 1996; Downer et al., in press, 2001).

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HANDBOOK OF RADIOPHARMACEUTICALS

The most extensive studies in breast cancer patients have been done with FES. In imaging with this agent, it has been shown that breast tumor uptake parallels the estrogen receptor content of tumors (McGuire et al., 1991), and, as expected, after a patient is placed on hormone therapy with the antiestrogen tamoxifen, subsequent images show that tumor uptake of FES is blocked, because this hormone or its high affinity metabolite, hydroxytamoxifen, occupy the estrogen receptors in the tumor (Dehdashti et al., 1995). More significantly, patients showing high initial uptake of FES proved to be much more likely to show a favorable response to hormone therapy (Dehdashti et al., 1999). Thus, as seen in Figure 21 tumor imaging with FES may prove to be a useful prognostic factor for the management of breast cancer patients.

Imaging of breast cancer in humans has also been done with other radioiodinated estrogens, 16aiodoestradiol and 11p-methoxy-17ct-iodovinylestradiol. Again, both primary and metastatic tumors could be detected with good sensitivity and selectivity, but so far, extensive correlations between imaging and clinical outcome have not been done (Kenady et al., 1993; Preston et al., 1990; Ribeiro-Barras et al., 1992; Rijks et al., 1997, 1998; Schober et al., 1990).

Figure 21. PET images of breast tumors obtained with the fluorine-18 labeled estrogen, 16a-[18F]fluoroestradiol (FES, left) and 2-[l8F]fluoro-2-deoxyglucose (FDG, right). In both cases, the image with FES is clearer than with FDG. SUV is standardized uptake value, and represents concentration over uniform distribution. Adapted from (Dehdashti et al., 1999); for details, see this reference. Reproduced with permission of Springer-Verlag GmbH & Co. KG.

IMAGING ANDROGEN RECEPTORS IN PROSTATE CANCER Androgen receptors are found in many prostate cancers, and these tumors typically show a favorable response to androgen ablative therapy (Petrylak, 1999). As summarized earlier in this chapter, a number of fluoroandrogens have been prepared and their tissue distribution studied in experimental animals; one of these, 16p-[18F]-5a-dihydrotestosterone (FDHT), has been studied in prostate cancer patients. When high doses of FDHT are administered, clear images of metastatic tumors are obtained (See Figure 22 below).

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

737

These studies are still in a preliminary stage, so the relationship between these images, androgen receptor levels and response to various therapies is not yet known.

Figure 22. PET reconstruction images of prostate tumors obtained with the metabolic agent, 2-[18F}fluoro~2~ deoxyglucose (FDG, top) and the fluorine-18 labeled androgen, l6p-[18F]fluoro-5a-dihydrotestosterone (pFDHT, bottom). Differential metabolic vs receptor-mediated uptake is noted at the sites indicated by the arrows, (Images provided by S. M. Larson, Memorial Sloan Kettering Cancer Center, reproduced with permission)

IMAGING OTHER NUCLEAR HORMONE RECEPTORS AND CANCER Progesterone receptor is a promising target for imaging breast cancer, because significant levels are found in many breast tumors, especially in those that are sensitive to hormone therapy (Clark & McGuire, 1983). In addition, progesterone receptor levels generally increase when a patient is placed on hormone therapy, rather than being blocked, as is the case with the estrogen receptor in patients treated with tamoxifen. Therefore, it is not surprising that considerable efforts have been made to develop progesterone receptor imaging agents, and the tissue distribution of many of these have been studied in rats.

21-[18F]fluoro-16<x-ethyl-19-

norprogesterone (FENP), a fluoroanalog of the potent progestin ORG2058, gave excellent receptor-mediated target tissue uptake in rats (Pomper et al, 1988).

However, in humans, this compound suffered rapid

deactivation through reduction of the C-20 ketone (Dehdashti et al., 1991; Verhagen et al., 1994).

A series of fluorine-18 labeled progesterone 16ct,17a~dioxolanes, based on a series of very potent oral progestins, were developed (Buckman et al., 1994, 1995; Kochanny et al., 1993), and the best of these called fluoro furanyl norprogesterone ketal (FFNP-ketal) is currently being evaluated in humans. It is

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HANDBOOK OF RADIOPHARMACEUTICALS

anticipated that the bulky ketal unit below the D-ring of this steroid will block or at least retard the deactivating reduction of the C-20 ketone (Verhagen et al., 1994).

Glucocorticoid receptors are found in lymphoid tissues and in lymphomas, so imaging based on this receptor might be useful for the diagnosis or staging of this cancer, or for monitoring or predicting the response to therapy. Various fluorine-18 labeled corticosteroids were developed, not for use in cancer, but for imaging the hypothalamus for the possible early diagnosis of Alzheimer's disease (Pomper et al., 1992). With some of the compounds, there was some evidence of receptor-mediated uptake in certain regions of the brain in rats; uptake in tumor systems was not investigated (Pomper et al., 1992). In general, the glucocorticoid receptor binds its ligands with lower affinity than do the sex steroid receptors (Pomper et al., 1992). Therefore, it is anticipated that imaging based on this receptor target will be challenging.

The peroxisome proliferator-activated receptor gamma (PPARy), a major regulator of lipid metabolism in some tissues, is also thought to play a role in regulating the growth of certain cancers (Sarraf et al., 1998). The tissue distribution of one fluorine-18 labeled analog of a high affinity PPARy ligand failed to show evidence of selective uptake in target tissues and tumors (Kim et al., 2001). The low liter of this receptor in tumors will make it a challenge to develop radiopharmaceuticals suitable for imaging of this target.

IMAGING SIGMA RECEPTORS IN CANCER The tissue distribution of a number of radiopharmaceuticals designed to be ligands for the sigma receptor show the hallmarks of receptor-mediated uptake: There is selective uptake in target tissues and tumors known to contain elevated levels of these receptors, with tumor to muscle ratios exceeding 7:1. Generally, a considerable fraction of this uptake can be blocked by an excess of unlabeled sigma receptor ligands (Choi et al., 2001; Dence et al., 1997; John et al, 1998, 1999b; Shiue et al., 2000; Waterhouse & Collier, 1997; Waterhouse et al., 1997a, 1997b, 1997c). So far, however, only limited imaging of tumors in humans have been done using these agents (Everaert et al., 1997).

CONCLUSION: FUTURE PROSPECTS FOR TUMOR RECEPTOR IMAGING IN THE ERA OF GENOME SCIENCES, TRANSGENIC ANIMAL MODELS, MICROIMAGING SYSTEMS, AND REGIONAL ISOTOPE PRODUCTION FACILITIES There are four recent, major technical advances that will provide an expanding array of opportunities and challenges for the development of radiopharmaceuticals for tumor receptor imaging.

RECEPTOR IMAGING OF TUMORS (NON-PEPTIDE)

739

First, through an analysis of the human genome, it is likely that new receptors will be identified and characterized. If it becomes evident that these new receptors are of importance in cancer, either by being linked to essential characteristics of the etiology of cancer or to tumor response to therapy, and if they bind small molecules, then they may function as imaging targets. It should be possible to develop radiopharmaceutials to assess, in vivo, the concentration and activity of these receptors in tumors.

Second, the development of transgenic and gene knockout technology has greatly facilitated gene functional studies in experimental animals, and this technology will certainly also facilitate the development of receptor imaging agents. Comparisons of the tissue distribution of a candidate imaging agent in normal animals with those in which the target receptor has either been knocked out or over expressed will permit a rapid assessment of the efficacy of the imaging agent directed at that target.

Third, the development of high resolution imaging methods suitable for small animals will enable information to be obtained from these valuable transgenic and knockout animals in an efficient and cost effective manner (Chatziioannou et al, 1999). In principle, quantitative and time course information obtained in a non-invasive manner by imaging a single animal will replace the need for the sacrifice of many animals to obtain the same information by tissue distribution studies. Along with these advantages will come new challenges, because the small volumes of target tissues and tumors that are sampled by microimaging devices will demand imaging agents of very high specific activity. Although many of the same agents that ultimately will be useful for imaging in humans should be adequate for imaging in small animals, the specific activity issue provides a further challenge for new developments in no-carrier-added radiosynthetic methodology.

Finally, a wider array of radionuclides of use in imaging (and, in some cases, also radiotherapy) is becoming available from regional production centers. Some of these radionuclides can be produced at the very high specific activity that is needed for tumor receptor imaging, particularly with small animals in the microimaging devices.

The development of radiopharmaceuticals that are suitable for imaging tumors through their (non-peptide) receptors has already progressed quite far (Welch et al, 1998). Presently, there are a few agents that provide excellent images of tumors based on their receptor content and thereby provide functional information on receptor concentration and activity that is proving to be of importance in making medical decisions. Nevertheless, there is very much more to be done in this field; many opportunities for further work are already clearly evident, and for the reasons outlined above, these opportunities are certain to expand greatly in the near future.

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HANDBOOK OF RADIOPHARMACEUTICALS

ACKNOWLEDGEMENTS The author is grateful to Al Wolf, whose work and career provided inspiration and encouragement to so many of us working in the field of radiopharmaceutical chemistry, and to Mike Welch, his coworkers and colleagues, with whom he has had a most productive collaboration for nearly 30 years. The author also wishes to thank many of his own coworkers for much of the work from his laboratory that is presented in this review, especially Kathryn Carlson, whose comments on the manuscript were particularly helpful. Grant support from the National Institutes of Health, the Department of Energy, and the American Health Assistance Foundation is also gratefully acknowledged. Hank Kung provided helpful information on sigma receptors prior to publication.

REFERENCES Al-Nabulsi I, Mach RH, Wang LM, Wallen CA, Keng PC, Sten K, Childers SR and Wheeler KT (1999) Effect of ploidy, recruitment, environmental factors, and tamoxifen treatment on the expression of sigma-2 receptors in proliferating and quiescent tumour cells. Brit. J. Can., 81,925-933. AH H, Rousseau J, Gantchev TG and van Lier JE (1993a) 2- and 4-Fluorinated 16a-[125I]iodoestradiol derivatives: synthesis and effect on estrogen receptor binding and receptor-mediated target tissue uptake. J. Med. Chem., 36, 4255-4263. AH H, Rousseau J, Ghaffari MA and van Lier JE (1988) Synthesis, receptor binding, and tissue distribution of (17a,20E)- and (17o,20Z)-21-[125I]iodo-19-norpregna-l,3,5(10),20-tetraene-3,17-diol.y. Med. Chem., 31, 1946-1950. AH H, Rousseau J and van Lier JE (1993b) 7a-Methyl- and 11ß-ethoxy-substitution of [125I]-16aiodoestradiol: effect on estrogen receptor-mediated target tissue uptake. J. Med. Chem., 36, 264–271. Allegra JC, Lippman ME, Thompson EB, Simon R, Barlock A, Green L, Huff KK, Do HMT and Aitken SC (1979) Distribution, frequency, and quantitative analysis of estrogen, progesterone, androgen, and glucocorticoid receptors in human breast cancer. Cancer Research, 39, 1447–1454. Anstead GM, Carlson KE and Katzenellenbogen JA (1997) The estradiol pharmacophore: ligand structureestrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids, 62, 268-303. Bergman J and Solin O (1997) Fluorine-18-labeled fluorine gas for synthesis of tracer molecules. Nucl. Med. Biol, 24, 677–683. Bonasera TA, ONeil JP, Xu M, Dobkin JA, Cutler PD, Lich LL, Choe YS, Katzenellenbogen JA and Welch MJ (1996) Preclinical evaluation of fluorine-18-labeled androgen receptor ligands in baboons. J. Nucl. Med., 37, 1009-1015. Bowen WD (2000) Sigma receptors: recent advances and new clinical potentials. Pharm. Acta Helv., 74, 211-218. Brandes SJ and Katzenellenbogen JA (1987) Fluorinated androgens and progestins: molecular probes for androgen and progesterone receptors with potential use in positron emission tomography. Mol. Pharm.,32,391-403.

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Brodack JW, Kilbourn MR, Welch MJ and Katzenellenbogen JA (1986) Application of robotics to radiopharmaceutical preparation: Controlled synthesis of fluorine-18 16a-fluoroestradiol-17ß, J. Nucl, Med,, 27,114–721. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J-A and Carlquist M (1997) Molecular basis of agonism and antagonism in the oestrogen receptor. Nature, 389, 753-758. Buckman BO, Bonasera TA, Kirschbaum KS, Welch MJ and Katzenellenbogen JA (1995) Fluorine-18labeled progestin 16a,17a-dioxolanes: Development of high-affinity ligands for the progesterone receptor with high in vivo target site selectivity. J. Med. Chem., 38, 328-337. Buckman BO, VanBrocklin HF, Dence CS, Bergmann SR, Welch MJ and Katzenellenbogen JA (3994) Synthesis and tissue biodistribution of [<j)-11C]palmitic acid. A novel PET imaging agent for cardiac fatty acid metabolism. J. Med. Chem., 37, 2481-2485. Chatziioannou AF, Cherry SR, Shao Y, Silverman RW, Meadors K, Farquhar TH, Pedarsani M and Phelps ME (1999) Performance evaluation of microPET: a high-resolution lutetium oxyorthosilicate PET scanner for animal imaging [see comments]. J. Nucl, Med., 40, 1164-75. Chesnut RW and Katzenellenbogen JA (1998) Synthesis of tetracoordinate dimethylgallium as potential iigands for the estrogen receptor. Organometallics, 17, 4889-4896. Chi DY, Kiesewetter DO, Katzenellenbogen JA, Kilbourn MR and Welch MJ (1986) Halofluorination of olefms: Elucidation of reaction characteristics and applications in labeling with the postronemitting radionuclide fluorine-18. J. Fluor. Chem., 31, 99–113. Chi DY, Lidstrom PJ, Choe YC, Bonasera TA, Welch MJ and Katzenellenbogen JA (1995) Bromo[18F]fluorination of cyclohexenes: A method for the preparation of [18F]fluorocyclohexanes. J, Fluor. Chem., 71, 143-147. Choe YS and Katzenellenbogen JA (1995) Synthesis of C-6 fluoroandrogens. Evaluation of ligands for tumor receptor imaging. Steroids, 60, 414–422. Choe YS, Lidstrom PJ, Chi DY, Bonasera TA, Welch MJ and Katzenellenbogen JA (1995) Synthesis of 1 lp[18F]fluoro-5a-dihydrotestosterone and 11ß-[18F]fluoro-19-nor-5a-dihydrotestosterone: Preparation via halofluorination-reduction, receptor binding, and tissue distribution. J. Med. Chem., 38, 816– 825. Choi SR, Yang B, Plossl K, Chumpradit S, Wey SP, Acton PD, Wheeler K, Mach RH and Kung HF (2001) Development of a Tc-99m labeled sigma-2 receptor-specific ligand as a potential breast tumor imaging agent. Nucl. Med. Biol., 28, 657-666. Clark GM and McGuire WL (1983) Progesterone receptors and human breast cancer. Breast Cancer Research & Treatment, 3, 157–163. Cummins CH (1993) Radiolabeled steroidal estrogens in cancer research. Steroids, 58, 245-259. Dehdashti F, Flanagan FL, Mortimer JE, Katzenellenbogen JA, Welch MJ and Siegel BA (1999) Positron emission tomographic assessment of "metabolic flare" to predict response of metastatic breast cancer to antiestrogen therapy. Euro. J. Nucl. Med., 26, 51–56.

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Dehdashti F, McGuire AH, Van Brocklin HF, Siegel BA, Andriole DP, Griffeth LK, Pomper MG, Katzenellenbogen JA and Welch MJ (1991) Assessment of 21-[18F]fluoro-16a-ethyl-19norprogesterone as a positron-emitting radiopharmaceutical for the detection of progestin receptors in human breast carcinomas. J. Nucl. Med., 32, 1532–1537. Dehdashti F, Mortimer JE, Siegel BA, Griffith LK, Bonasera TJ, Fusselman MJ, Detert DD, Cutler PD, Katzenellenbogen JA and Welch MJ (1995) Positron tomographic assessment of estrogen receptors in breast cancer: comparison with FDG-PET and in vitro receptor assays. J. Nucl. Med., 36, 1766– 1774. Dence CS, John CS, Bowen WD and Welch MJ (1997) Synthesis and evaluation of [I8F] labeled benzamides: high affinity sigma receptor ligands for PET imaging. Nucl. Med. Biol., 24, 333–40. Dence CS, Napolitano E, Katzenellenbogen JA and Welch MJ (1996) Carbon-11 -labeled estrogens as potential imaging agents for breast tumors. Nucl. Med. Biol., 23, 491–496. DeSombre ER, Pribish J and Hughes A (1995) Comparison of the distribution of radioiodinated di- and trihydroxyphenylethylene estrogens in the immature female rat. Nucl. Med. Biol., 33, 679–687. DiZio JP, Anderson CJ, Davison A, Ehrhardt GJ, Carlson KE, Welch MJ and Katzenellenbogen JA (1992) Technetium- and rhenium-labeled progestins: Synthesis, receptor binding and in vivo distribution of an 11ß-substituted progestin labeled with technetium-99 and rhenium-186. J. Nucl. Med., 33, 558569. DiZio JP, Fiaschi R, Davison A, Jones AG and Katzenellenbogen JA (1991) Progestin-rhenium complexes: Metal-labeled steroids with high receptor binding affinity, potential receptor-directed agents for diagnostic imaging or therapy. Bioconj. Chem., 2, 353-366. Downer JB, Jones LA, Engelbach JA, Lich LL, Mao W, Carlson KE, Katzenellenbogen JA and Welch MJ Comparison of animal models for the evaluation of radiolabeled androgens. Nucl. Med. Biol., 6, 613-626. Endo Y, lijima T, Yamakoshi Y, Yamaguchi M, Fukasawa H and Shudo K (1999) Potent estrogenic agonists bearing dicarb-c/oso-dodecarborane as a hydrophobic pharmacophore. J. Med. Chem., 42, 1501– 1504. Everaert H, Flamen P, Franken PR, Verhaeghe W and Bossuyt A (1997) Sigma-receptor imaging by means of I23I-IDAB scintigraphy: clinical application in melanoma and non-small cell lung cancer. Anticancer Research, 17, 1577–1582. French AN, Napolitano E, VanBrocklin HF, Hanson RN, Welch MJ and Katzenellenbogen JA (1993a) Synthesis, radiolabeling and tissue distribution of 1 ip-fluoroalkyl- and 11ß-fluoroalkoxysubstituted estrogens: Target tissue uptake selectivity and defluorination of a homologous series of fluorine-18-labeled estrogens. Nucl. Med. Biol., 20, 31–47. French AN, Wilson SR, Welch MJ and Katzenellenbogen JA (1993b) A synthesis of 7a-substituted estradiols: Synthesis and biological evaluation of a 7a-pentyl-substituted BODIPY fluorescent conjugate and a fluorine-18-labeled 7a-pentylestradiol analog. Steroids, 58, 157-169. Frost JJ (1982) In Receptor-binding radiotracers, Vol. II (Ed, Wiliam C. Eckelman, P. D.) CRC Press, Boca Raton, Florida, pp. 25-39.

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Waterhouse RN and Collier TL (1997) In vivo evaluation of [F-18]-l-(3-Fluoropropyl)-4-(4Cyanophenoxymethyl)piperidine - a selective sigma-1 receptor radioligand for PET. Nucl. Med. Biol., 24, 127-134. Waterhouse RN, Mardon K, Giles KM, Collier TL and OBrien JC (1997b) Halogenated 4(phenoxymethyl)piperidines as potential radiolabeled probes for sigma-1 receptors: in vivo evaluation of [123I]-1- (iodopropen-2-yl)-4-[(4-cyanophenoxy)methyl]pip eri dine. J. Med. Chem., 40, 1657-67. Waterhouse RN, Mardon K and O'Brien JC (1997c) Synthesis and preliminary evaluation of [123I]1-(4cyanobenzyl)-4- [[(trans-iodopropen-2-yl)oxy]methyl]piperidine: a novel high affinity sigma receptor radioligand for SPECT. Nucl. Med. Biol., 24, 45-51. Weatherman RV, Fletterick RJ and Scanlan TS (1999) Nuclear-receptor ligands and ligand-binding domains. Annual Review of Biochemistry, 68,559-581. Welch MJ, McCarthy DW, Lewis MR, Shefer RE, Klinkowstein RE and Hughey BJ (1998) Production of radionuclides for therapy using small cyclotrons. In 215th ACS National Meeting American Chemical Society, Washington, D. C, Dallas, TX. Wheeler KT, Wang LM, Wallen CA, Childers SR, Cline JM, Keng PC and Mach RH (2000) Sigma-2 receptors as a biomarker of proliferation in solid tumours. Brit. J. Can., 82, 1223–1232. Wilson AA, Dannals RF, Ravert HT, Sonders MS, Weber E and Wagner HN (1991) Radiosynthesis of sigma receptor ligands for positron emission tomography: "C- and 18F-labeled guanidines. J. Med. Chem., 34, 1867–1870. Wolfe SA and De Souza EB (1994) Role of sigma binding sites in the modulation of endocrine and immune functions. In Sigma Receptors; Neuroscience Perspectives Series Academic Press, Horcourt Brace & Co., New York, pp. 225-242. Wurtz J-M, Bourguet W, Renaud J-P, Vivat V, Chambon P, Moras D and Gronemeyer H (1996) A canonical structure for the ligand-binding domain of nuclear receptors. Nature Structural Biology, 3, 87-94. Wust F, Carlson KE, Katzenellenbogen JA, Spies H and Johannsen B (1998) Synthesis and binding affinities of new 17a-substituted estradiol-rhenium "n+1" mixed-ligand and thioethercarbonyl complexes. Steroids, 63, 665-671. Wiist F, Scheller D, Spies H and Johannsen B (1997) Synthesis of oxorhenium(V)complexes derived from 7a-functionalized testosterone: first rhenium-containing testosterone derivatives. Euro. J. Inorg. Chem., 789-793. Wiist F, Spies H and Johannsen B (19%) Synthesis of "3+1" mixed-ligand oxorhenium(V) complexes containing modified 3,17p-estradiol. Bioorg. Med. Chem. Letters, 6, 2729-2734. Young PW, Buckle DR, Cantello BCC, Chapman H, Clapham JC, Coyle PJ, Haigh D, Hindley RM, Holder JC, Kallender H, Latter AJ, Lawrie KWM, Mossakowska D, Murphy GJ, Cox LR and Smith SA (1998) Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (Brl49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferatoractivated receptor gamma. J. Pharm. Exp. Thera., 284, 751–759.

26. PULMONARY FUNCTION IMAGING WITH PET RADIOPHARMACEUTICALS P.H. ELSINGA AND W. VAALBURG Groningen University Hospital, PET-center, P.O. Box 30001, 9700 RB Groningen, The Netherlands

INTRODUCTION The lung is a vital organ of the human body with its main function the exchange of oxygen from inhaled air with carbon dioxide from blood and to expire the exchanged CO2. The actual gas exchange takes place in the alveoli through an 0.1 |im thick membrane. The O2 containing inhaled air is transported to the alveoli through the respiratory tract. The upper airway tract branches into two bronchi which in turn branch into bronchioles and terminate in alveoli. Deoxygenated CO2-rich blood flows from the right ventricle of the heart, through the pulmonary artery, to the lungs. In the lungs the pulmonary artery branches into arterioles with diameters of 25–35 un. These arterioles in turn branch into capillaries with an average diameter of 8 um, just wide enough to allow red blood cells to pass. Each alveolus is surrounded by about 1000 capillaries. Through the alveolar membrane the blood is brought into contact with inhaled air allowing 02 to diffuse into the blood and CO2 into the air. The air flow through the lung is called ventilation and the blood flow through the lung perfusion. Normal ventilation and perfusion are essential for an effective alveolar gas exchange. Lung function in the sense of gas exchange is determined by the regional matching of ventilation and perfusion. Lung function is also controlled by the endothelial receptor systems, enzyme functions and metabolic processes. Neural control of airway smooth muscle resulting in contraction and relaxation is an important determinant of airway caliber in health and disease (Barnes, 1997). Several neurotransmitters in airway nerves and receptors on airway smooth muscle cells have been identified. Acetylcholine and (nor)epinephrine act on muscarinic receptors (bronchocontraction) and on a- (bronchoconstriction) and p*-adrenoceptors (bronchodilatation) respectively. Neuropeptides can act inhibitory (vasoactive intestinal polypeptide (VIP) and nitric oxide) or excitatory (substance P and neurokinin A). It is increasingly recognized that a single transmitter may act on several types of receptors. Ventilation and perfusion studies are indicated for embolic as well as for non-embolic diseases such as chronic obstructive pulmonary diseases (COPD), asthma and cystic fibrosis. Perfusion studies are useful if e.g., a partial or complete obstruction of the A. pulmonalis or a left-right shunt is suspected. The methods are also used to estimate lung function before and after surgery or radiotherapy. However, the main clinical application is for the diagnosis of pulmonary thromboembolism. Neural mechanisms may be involved in the pathophysiology of asthma or COPD. Endothelial dysfunction and inflammation may also affect lung function parameters. There is also a close relationship between inflammation and neural responses in the airways. Handbook of Radiopharmaceuticals. Edited by M. J Welch and C. S, Redvanly. ©2003 John Wiley & Sons, Ltd

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A non-invasive in vivo diagnostic tool to investigate the afore-mentioned abnormalities is of great clinical interest. Nuclear medicine techniques like PET and SPECT are useful imaging modalities for this purpose. Imaging of the lung is accompanied by significant problems (Schuster, 1998). - The lung is an air-containing organ. The PET/SPECT signal may change not only because of changes in radioactivity uptake, but also as a result of changing lung volume. - The lung also contains large amounts of blood, so a proper signal originating from lung tissue only is hard to measure. - Respiratory motion is another problem in lung studies. Not only for diagnostic purposes but also for research and better understanding of normal physiology and pathology, radioactive tracer methods are applied. For the preparation and application of single photon radiopharmaceuticals for measuring lung parameters, adequate reviews are available in the literature and in text books (Chan, 1993; Dolovich, 2001). The evaluation of lung function by PET has recently been reviewed (Rhodes & Hughes, 1995; Schuster, 1998). PET research of pulmonary function is mainly focussed on ventilation/perfusion and neural control/metabolism of smooth muscle. Other reports on a variety of subjects of much less attention or interest will not be dealt with in this chapter. This chapter gives an overview of the positron emitting radioactive gases for lung function studies and the current possibilities and radioligands to assess receptor parameters and metabolism of lung tissue.

PERFUSION/VENTILATION GASES For routine clinical application, the single photon emitting gases 127Xe,133Xe and 8lmKr are most widely used to measure the ventilation/perfusion ratio. These radiotracers are commercially available either as gas, as injectable solution, or as generator system. A disadvantage of xenon is its relatively high solubility in blood and tissue which introduces errors in the measured perfusion and ventilation (van der Mark et al., 1984). Besides its solubility, l33Xe has the disadvantage of a low photon energy of 81 keV which makes the evaluation of deeper lung areas and the use of rotating SPECT systems difficult. As an alternative, 81mKr can be used. For ventilation studies the tracer can be applied as gas and for perfusion studies as a saline solution. For gamma cameras the radionuclide has a convenient photon energy of 180 keV. The very short half-life of 13 seconds makes the distribution logistics complicated but it has the advantage that measurements can be repeated immediately. The gas is available from a 8l Rb/ 8lm Kr generator. The mother radionuclide of this generator system has a half-life of 4.7 hours, which allows distribution.

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Table 1. Solubility of some gases in water Gas

Isotope

Solubility (cm 3 /100gH 2 O)

T (°

Nitrogen Neon Krypton Xenon

U

1.42 1.47 6.0 11.9

40 20 25 25

19

N Ne

81rnKr 133

Xe

Nitrogen and neon have a very low solubility in tissue. From Table 1 it can be concluded that the solubilities of 19Ne and 13N2 are nearly ten times lower than that of xenon. The low solubility of these tracers make them the radiopharmaceuticals of choice to measure pulmonary ventilation and perfusion. Another advantage of these positron emitting gases is that in combination with a modern PET camera (Hughes et al, 1987), cross sectional images of the lungs can be obtained with a much better resolution than with a gamma camera or SPECT system. Moreover, the organ distribution of positron emitting radionuclides can be measured quantitatively. However, 19Ne and 13N2 have limitations because of the half-lives of 17 seconds and 10 minutes, respectively. The short half-lives reduce the availability. The radionuclides have to be produced on site. For this purpose, most often a medical cyclotron is used. 19

Ne as gas is applied for the measurement of the ventilation and 13N2 for ventilation and perfusion studies. The clinical value of these tracers is not yet been established. Also H215O has been used for lung perfusion measurements. Only institutions with an on site cyclotron and a PET camera can apply the tracers. However, mis situation is improving with an increasing number of installed medical cyclotrons and PET cameras. NEON-19 Because of the low solubility of neon its distribution volume essentially represents the ventilated lung. It can be regarded as the positron emitting equivalent of 81mKr including its advantage that studies can be repeated immediately, because of the short half-life. Unfortunately, because of the maximum positron energy of 2.24 Me V the positron range in tissue is rather high. This high range can affect the spatial resolution, depending on the positron imaging device used. Neon-19 can be produced via the 19F(p,n)19Ne and the 16O(ct,n)19Ne reactions. The most effective production reaction is the a-bombardment of a continuous O2 flow (Crouzel, 1980). The only chemical impurity in the irradiated O2 gas is ozone, which can easily be removed by a manganese dioxide filter. Unfortunately, most medical cyclotrons installed in hospitals today are not capable of accelerating a-particles. The l9Ne produced is transported through a flow system to the patient investigation room. While the patient is breathing a mixture of 19Ne and air, data acquisition is performed. It has been shown that in COPD patients ventilation heterogeneities can be detected tomographically.

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NITROGEN-13 LABELLED MOLECULAR NITROGEN From a physiological point of view I3N- gas is the most appropriate radioactive gas. For ventilation studies I3

N2 is applied as molecular gas and for perfusion studies as13N2 in physiological saline. Several authors

describe the advantage of 13N2 over other inert gases for more precise regional ventilation or perfusion studies, including models to quantitate these parameters (McKenzie & Fitzpatrick, 1981; Ahluwalia et al., 1981; Senda et al., 1986; Murata et al., 1986). The short half-life of I3N also necessitates production of the radioactivity on site. For this purpose several nuclear reactions are applied. The most important reactions are the 12C(d,n)l3N, l3C(p,n)13N and l6O(p,a)13N reactions. For the nuclear reaction on carbon, either CO2 (Murata et al., 1986), graphite or activated charcoal are used as target materials. Water as well as CO2 is used as targets for the (p,a) reaction. Disadvantages of the carbon-based production methods are that targetry is relatively complex and that only injectable solutions with a relatively low radioactivity per ml physiological saline can be obtained. All reactions mentioned can be used to prepare the radioactivity continuously as well as batchwise. Production systems based on the irradiation of carbon, including target design, target performance and flow systems are described in detail (Clark & Buckingham, 1975). A more simple production method of 13N2 gas as well as l3N2-solutions is based on the l6O(p,a)13N reaction and water as target material (Vaalburg et al., 1981). The main advantages of this approach are simple targetry, high absolute yield of 13N and a very high amount of radioactivity per ml gas or per ml injectable 13N2solution. The batchwise production allows transport of the radioactivity without a flow system. The method is based on the oxidation of 13NH3 by sodium hypobromite. 2

13

NH3 +

3 NaOBr

>

13

N2 + 3 NaBr

+

3 H2O

The [13N]ammonia is prepared by irradiation of water with protons followed by the reduction of I3NO3 and 13 NO2 which are formed in the target during bombardment. When the labeled gas is produced for ventilation studies, [l3N]ammonia is collected in a vial containing carrier NH4C1. For perfusion studies the 13N2 is generated in solution under sterile conditions. After destroying the excess NaOBr with ascorbic acid, the solution is ready for intravenous injection. PARTICLES Instead of using inert radiolabelled molecular gases, ventilation can also be measured with radiolabelled aerosols such as [99MTc]-diethylene triamine pentaacetic acid (DTPA), [99mTel-labelled carbon particles or Technegas. However, comparisons of radioactive inert gases and aerosols did not result in major diagnostic differences (Lowe & Sostman, 1998; Van der Wall & Magnussen, 1998; Tagil et al., 2000). For perfusion measurements, radiolabelled macroaggregated albumin is used. After intravenous injection of the albumin aggregates the radiolabelled particles are trapped in perfused areas of the lungs because the size of the particles is too large to pass the pulmonary capillaries. The number of particles trapped is a measure of the perfusion in that area. [99mTcl-macroaggregated albumin is clinically the most frequently applied radiopharmaceutical for perfusion measurement. If the advantages of PET with its high resolution and its cross sectional quantitative capabilities are required,

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macro- and microaggregates can be labelled with 68Ga (Even & Green, 1989; Wagner & Welch, 1979) or 11 C (Turton et al, 1984). For the 68Ga-labelling standard techniques are used. Gallium-68 is available as 68

Ge/68Ga generator (Loch et al., 1980). Unfortunately these generators are expensive and not widely

available. Carbon-11 methyl-labelled albumin has been prepared by [11C]-memylation of commercially available mieroor maeroaggregates. Turton et al. showed that the [HC]albumin particles are highly stable in vivo and that [11C]albumin aggregates may be used as reference blood flow marker. In comparison to the positron emitting radioactive gases, the relatively long half-lives of 68Ga and "C make sequential studies more difficult. At present these tracers are only used in research settings.

NEURAL CONTROL AND METABOLISM Most attention in PET-research has been focused on P-adrenoceptors. Several groups have worked on this topic. Only a few papers appeared on other pulmonary receptors, whereas research on neuropeptides emerged recently. A few radioligands in the field of neuropeptide action have been prepared, but not evaluated. BETA-ADRENOCEPTORS In lung, P-adrenoceptors are involved in numerous functions like airway smooth muscle relaxation, mucus secretion, ion transport across airway epithelium and permeability of pulmonary blood vessels. Stimulation of p2-adrenoceptors in the lungs by p2-agonists results in activation of adenylate cyclase via the stimulatory Gs protein followed by the formation of the second messenger, cyclic AMP. Cyclic AMP accumulation then leads via a cascade of processes to relaxation of smooth muscle cells in the walls of the airways. Such relaxation seems to be impaired in humans with pulmonary dysfunction, e.g., in patients suffering from asthma or COPD. p-Adrenoceptor density in human peripheral lung is normally 100–130 fmol/mg protein and the ratio of the pt: p2 subtypes is about 30:70 (Brodde, 1991). Several p-adrenoceptor ligands have been labeled with positron emitting isotopes to measure P-adrenoceptors in lung with PET. When injected intravenously, the uptake of radiolabeled P-adrenoceptor ligands in the airways reflects binding to alveolar sites rather than binding to receptors on smooth muscle cells. Alveolar Padrenoceptors are not directly involved in the pathophysiology of asthma. Therefore, it seems worthwhile to perform PET studies with inhaled P-adrenoceptor ligands. Inhalation may result in the selective labeling of airway rather than alveolar P-adrenoceptors. (S)-[11C]CGP 12177 (S)-[11'C]CGP 12177 is an established radioligand producing high-contrast PET-images. The radioligand has been used to determine pulmonary p-adrenoceptor density in humans using a two-injection protocol. Pulmonary beta-adrenoceptor density measured by PET after i.v. injection of [11C]CGP 12177 correlated quite closely with in vitro binding assays (Qing et al., 1996). In another study, the downregulation of pulmonary P-adrenoceptors during 2 weeks of treatment with albuterol (oral and inhaled) was assessed. Pulmonary P-adrenoceptor density fell by 22%, while

P-adrenoceptor numbers on the surface of

mononuclear leukocytes dropped by 42% (Hayes et al, 1996). The changes in pulmonary and leukocyte Padrenoceptors were significantly correlated. A reduction in the bronchodilator response to inhaled salbutamol

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was also observed. In contrast, acute infusion of salbutamol did not affect P-adrenoceptor density in human lungs. Asthmatic subjects showed similar pulmonary P-adrenoceptor densities as aged-matched healthy controls (10.3 versus 10.9 pmol/g tissue). These data were interpreted as indicating the absence of a primary Padrenergic deficit in asthma. However, it should be noted that the employed imaging technique visualizes all P-adrenoceptors (iv injection) and not just p-adrenoceptors in the airways. One may speculate that changes in the relatively small population of airway P-adrenoceptors were not detected in these studies because of the very strong signal arising from the alveoli. Interestingly and as yet unexplained, an inverse relationship was observed between pulmonary function and pulmonary p-receptor density in asthmatic subjects.

"qphosgene

11 '

Figure 1. Synthesis of (S)-[11C]CGP 12177 from [11C]phosgene. (S)-[11C]CGP 12177 is produced by reaction of [11C]phosgene with the appropriate (S)-diamine precursor (Figure 1) in high radiochemical yield (Aigbirhio el al., 1992;Boullais et al, 1986; Brady et al., 1991). Unfortunately, several PET centers reported variable and usually very low specific activities. The troublesome and laborious radiolabeling via [11C]phosgene is an important disadvantage to the application of [11C]CGP 12177 for clinical studies and prevents widespread use of this radioligand. (S)-[11C]CGP 12388 Because of the problems involved in the routine production of (S)-[11C]CGP 12177, it was considered worthwhile to develop a hydrophilic P-adrenoceptor ligand for clinical use. Recently, (S)-CGP 12388 was selected as a suitable candidate (Elsinga et al., 1997). CGP 12388 is the isopropyl analog of CGP 12177. In vitro experiments have indicated that racemic CGP 12388 is almost equally potent as racemic CGP 12177 (unpublished results of Ciba-Geigy). In vivo blocking experiments have indicated that (S)-[11C]CGP 12388 binds to p r and p2-adrenoceptors. CGP 20712A (Pi-selective) diminished myocardial but not pulmonary binding of the radioligand (van Waarde et al., 1998). In contrast, ICI 118, 551 ( Pj-selective) reduced uptake in the lungs without affecting binding in the heart. The metabolic rate of (S)-[11'C]CGP 12388 in male Wistar rats was similar to that of (S)[11CJCGP 12177. The fraction of plasma radioactivity representing parent compound decreased from 99% at time zero to 61 % at 40 min post injection. Radiochromatograms of tissue extracts made 60 min post injection indicated that heart and lungs contain mainly unchanged (S)-[11C]CGP 12388. (S)-[11C]CGP 12388 has been evaluated in healthy human volunteers (Elsinga et al., 1999). In the control studies, the myocardial left ventricle, peripheral lung, liver and spleen were clearly visible (Figure 2). After administration of pindolol, the left ventricle, lung and spleen were no longer visible, whereas the uptake in liver was unchanged. After ingestion of pindolol, the maximal tissue uptake decreased, whereas the washout

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rate was increased. Pindolol reduced the uptake of radioactivity in heart, lung and spleen by 76%, 68% and 77% respectively at 60 min post injection.

Figure 2. PET image of the human thorax, acquired with (S)-[11C]-CGP 12388. A transaxial cross-section of the thorax is displayed containing the data summed over the last 14 frames. The left hand image is the control study and the right-hand image is obtained after blocking with pindolol. The lungs are clearly visualized.

(S)-[11C]CGP 12388 was labeled via a one-pot procedure using 2-[11C]acetone (Figure 3)(Elsinga et al, 1997). The radiochemical yield was 15-20% EOB. Total synthesis time is 40 min with specific activities of 20,000-40,000 GBq/mmol. This synthetic procedure is easily performed and therefore more suitable for clinical use than the multistep synthesis of (S)-[11C]CGP 12177 from [11C]phosgene.

11

NH

C] acetone „

O

NH

NH

Figure 3. Synthesis of (S)-[11C]CGP 12388 by reductive alkylation with [11C]acetone.

Because of ease in preparation and an in vivo behavior comparable to (S)-[11C]CGP 12177, (S)-[11C]CGP 12388 seems a promising tracer for in vivo studies of peripheral p-adrenoceptors. Further evaluation in humans should determine whether CGP 12388 is a useful radiopharmaceutical for clinical PET. (SM18F]fluorocarazoIol Fluorocarazolol is a high-affinity, lipophilic (logP 2.2) p-adrenoceptor ligand, which has been evaluated by several groups (Visser et al., 1997a; Zheng et al., 1994).

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In PET images of male Wistar rats and lambs, the lungs were clearly visible and pulmonary uptake of radioactivity was decreased (>90%) after pretreatment of the animals with propranolol. An animal model of asthma using rabbits was developed (Hakernarson et al., 1997) and PET studies with (S)-[l8F]fluorocarazolol were performed. In these preliminary experiments the absolute uptake of radioactivity in the lungs of the asthmatic animals tended to be 40% lower man in the control studies. PET scans after i.v. injection of high specific activity (S)-[l8F]fluorocarazolol in human volunteers clearly showed B-adrenoceptors in both lung and heart, and in specific brain areas. The radioactivity uptake was blocked by pindolol pretreatment. Metabolite analyses of [18F]fhiorocarazolol showed a rapid appearance of polar metabolites in plasma, while at 60 min post injection 92% and 82% of the total radioactivity in lung and heart remained unchanged (S)[l8F]fluorocarazolol. Extensive toxicity studies with (S)-fluorocarazolol showed a positive Ames Test (mutagenicity in bacterial strains) (Doze et al, 2000). (SMl8F]fluorocarazolol was prepared by reductive alkylation of [l8F]fluoroacetone with (S)-desisopropylcarazolol (Figure 4). Radiochemical yields were 5% (EOB) with a synthesis time of 180 min and specific activities of > 80,000 GBq/mmol. (S)-[11C]Carazolol Like fluorocarazolol, carazolol is lipophilic and has high affinity for the fJ-adrenoceptor. Lung studies have

r18. 18r ['°F]fluorocarazolol: R, = IO F

[11C]carazolol: *C =11C; RT = H Figure 4. Synthesis of 18F- and 11C-labelled (S)-carazolol from the corresponding (S)-desisopropyl precursor.

been carried out (Berridge et al., 1994) in mice and pigs. Tracer uptake in lung was measured by PET as a function of time. Receptors were blocked with propranolol (to 38% at 60 min post injection) and different doses of ICI118, 551 to estimate specific binding. Also, a large difference was observed when (S)-carazolol was compared to the biologically inactive (R)-isomer (to 10% of (S)-binding at 60 min post injection). PET images in untreated pigs were of good quality and contrast. Specific receptor binding was observed. Metabolite analysis demonstrated 20% metabolites in blood at 6 min post injection. The fraction remained constant up to 2 hours after injection.

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(S)–[11C]Carazolol is produced from [11C]acetone and the corresponding (S)-desisopropyl compound by reductive alkylation (Figure 4) (Berridge et al., 1992). The radiochemical yield was 12% EOB with a synthesis time of 30 min. Specific activities ranged from 80,000–100,000 GBq/mmol.

[11C]-Formoterol To examine whether it is possible to image pulmonary p2-adrenoceptors in vivo with a radiolabeled agonist, [11'CHormoterol was synthesized (Visser et al., 1998). Formoterol displays a high affinity (KD = 1.05 ± 0,17 nM) in human lung membranes (Mak et al., 1994) and selectivity (P 2 : ($1=90:10)for the p2-subtype (Roux et al., 1996). Moreover, as a drug it has been used extensively in the clinic because it reduces asthmatic symptoms significantly and it causes a long lasting bronchodilatation in man when administered by inhalation. Formoterol has been labeled with [3H] and is used in in vitro binding studies. In human and guinea pig lung membranes [3H]formoterol binds to only one class of receptors. It was shown that formoterol selectively labels the high affinity state of the p-adrenoceptors. The high affinity state of the receptor is believed to bring about the signal that evokes the biological effect. Selective labeling of the high affinity state can be acquired only with agonist ligands. Such data can be of clinical interest because the fraction of receptors in the high affinity state may be altered by disease or after treatment. The efficacy of agonist drugs may be affected by alterations of the fraction of receptors in the high affinity state. Labeling of formoterol with a positron emitter was, therefore, highly interesting. In a PET study with [11C]formoterol in male Wistar rats, lungs were clearly visible. After pretreatment of the rats with propranolol, the lungs could no longer be seen. Biodistribution studies in male Wistar rats, either untreated or predosed with propranolol, showed significant specific binding in tissues known to contain p2adrenoceptors (lungs, spleen, heart). Uptake in these target organs was blocked by the p2-adrenoceptor antagonist ICI 118,551 and the non-selective p-adrenoceptor agonist isoprenaline but not by the P,adrenoceptor antagonist CGP 20712A. These results are consistent with the p2-selectivity of formoterol and they show that it is possible to image p-adrenoceptors with a radiolabeled agonist. Formoterol (a mixture of (S,S)- and (R,R)-isomers) was labeled via reaction of a benzyl-protected precursor with [11C]CH3I (Figure 5). Subsequent deprotection with Pd/C and H2 yielded [nC]formoterol in 5–15% BOB and the specific activity ranged from 5-22 TBq/mmol, 60–70 min EOB.

HO' Figure 5. Chemical structure of [11CJformoterol.

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MUSCARINIC RECEPTORS Muscarinic receptors are widely distributed in lung and are found on submucosal glands, airway ganglia, smooth muscle of large and small airways and alveolar walls (Barnes, 1993; Mak & Barnes, 1990). Mireceptors are located on submucosal glands and alveolar walls in the peripheral lung. M2-receptors act as autoreceptors on post-ganglionic cholinergic nerves and inhibit acetylcholine release. The M3-subtype on the surface of the tracheal smooth muscle fibres, the bronchi, and the bronchioles, mediates muscle contraction. Pulmonary mucus secretion is stimulated via the M1- and M3-subtypes in submucosal glands. In the rat lung, the M2-subtype predominates (80-90%), whereas in humans up to 70% of the receptors are of the M1 subtype and the remaining of the M3-subtype. Only a very small portion is of the M2-subrype. The density of muscarinic receptors in rat peripheral lung ranges from 21-35 fmol/mg protein. In human lung a receptor density of 21-165 fmol/mg protein is reported (Mak, 1990). Muscarinic receptors play an important role in bronchoconstriction and, therefore, in the pathophysiology of asthma and COPD. Increased muscarinic cholinergic activity may occur through an increase in receptor density or affinity, or through an increase in the efficacy of receptor/signal transduction coupling. Only a few PET studies of muscarinic receptors in the airways have been reported. Research on these receptors focussed on brain and heart. A major obstacle in the quantification of muscarinic receptors in the lungs is the relatively low density. A few radioligands have been prepared and only one radioligand has been evaluated in man to measure muscarinic receptors in lung. N-[11C]-methyl-piperidin-4-yl-2-cyclohexyl-2-hydroxy-2-phenylacetate ((R)-[11C]VC-002) was evaluated in

Figure 6. PET-images of the lungs of a healthy volunteer acquired with (R)-[11C]-VC-002 before and after blockade. Transaxial cross-sections are shown.

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healthy human volunteers (Visser et al., 1999). The radioligand was administered on two separate days. The first injection was without pretreatment; prior to the second injection the anticholinergic compound glycopyrronium bromide was injected to block the receptors. In PET images of the thorax (Figure 6), the lungs were clearly visible. Pulmonary radioactivity uptake was reduced to 30% at 60 min post injection after blocking with glycopyrronium bromide. [11C]VC-002 was rapidly cleared from plasma and metabolism was negligible during the PET investigation. (R)-[11C]VC-002 was prepared by [11C]methylation of the corresponding desmethyl precursor (Visser et al., 1997b) as shown in Figure 7. (R)-[11 C] VC-002 was produced with a radiochemical yield of 40–60% (EOB), a specific activity of >11000 GBq/mmol and synthesis time of 40 min.

Figure 7. Synthesis of (R)-[11C]VC-002 by [11C]methylation.

ANGIOTENSIN CONVERTING ENZYME INHIBITOR Angiotensin-converting enzyme inhibitors are potent vasodilators acting by inhibition of production of the vasoconstrictor angiotensin II (Schneeweiss, 1988). They are used for treatment of systemic hypertension, congestive heart failure and primary pulmonary hypertension (PPH). The ACE-inhibitor captopril was radiolabeled with fluorine-18 to study the feasibility of probing the angiotensin-converting enzyme (ACE) in vivo using PET (Qing et al., 2000). The resulting 4-cis-[l8F]fluorocaptopril was administered to rats. High uptakes at 30 min post injection were observed in structures known to have high ACE-densities, like lung, kidney and aorta. Saturation experiments with 'cold' fluorocaptopril showed ED50 values in lung of >5ug/kg body weight and in kidney of 1 Mg/kg body weight. The binding was saturable with the expected capacity. In humans, a displaceable uptake in lung and kidney was measured. In another study, PET was used to determine the dose of ACE inhibitor required to specifically block pulmonary ACE in humans. The combined forward rate constant (CFRC) for [l8F]fluorocaptopril, which is proportional to the ACE-density in the lung, was determined. In healthy human volunteers, the CFRC decreased with 84% after ingestion of enalapril on a daily base. In patients with PPH, CFRC decreased with 76% after 1 week enalapril treatment. It was concluded that the total density of pulmonary ACE was significantly reduced in PPH and that only low doses of ACE inhibitors are needed to block the effects of ACE on vascular remodeling in PPH. 4-Cis-[18F]fluorocaptopril is prepared by the fluorination of the corresponding triflate (Hwang et al., 1991), followed by alkaline hydrolysis. After a synthesis time of 60 min, a radiochemical yield of 24% (EOB) and specific activities of >11,000 GBq/mmol were obtained.

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AMINE METABOLISM AND PHARMACOKINETICS The single pass extraction of many amines by the lung is very effective. A reduction of amine uptake has been observed in acute pulmonary disease characterized by endothelial dysfunction. The first pass extraction can be measured with PET. Several papers have been reported on this topic. The fraction of serotonin extracted from a single passage through the lungs is being used as an early indicator of lung endothelial damage. The double indicator diffusion principle was applied using a positron camera, with [11C]serotonin as the substrate, and with [11CJCO-erythrocytes as the vascular marker (Coates et al., 1991). Time-activity curves were recorded after a bolus injection of the first vascular marker [11C]COerythrocytes and 10 min later injection of 11CJserotonin. A second uptake measurement was made after imipramine was infused intravenously. In three normal volunteers, the single-pass uptake of [11CJserotonin was 64%. This decreased in all subjects to a mean of 54% after imipramine treatment. The rate of lung washout of 11C was also significantly prolonged after administration of imipramine. This noninvasive technique can be used to measure lung serotonin uptake to detect early changes in a variety of conditions that alter the integrity of the pulmonary endothelium. In patients, the uptake of [11CJchlorpromazine (CPZ) was measured to evaluate the nonrespiratory function (Syrota et al., 1981). A multiple-indicator dilution technique was used with external detection. Intravenous bolus injection of [11CJCPZ was followed by [113mIn]transferrin as an intravascular reference molecule. The initial extraction was 90% in four normal subjects and 64% in six patients with chronic obstructive lung disease (COLD). The concentration achieved in the infected tissue is of fundamental importance in the use of antibiotics. [11C]Erythromycin was used to compare local concentrations of this antibiotic in the pneumonic and unaffected lungs of five patients with lobar pneumonia (Wollmer et al., 1982). The mean extravascular concentrations observed during the first hour after intravenous injection of erythromycin lactobionate were similar in the pneumonic lung and the unaffected lung. An effective concentration of erythromycin was reached in the pneumonic lung within 10 min after injection and was maintained throughout the period of measurement (60 min). CONCLUSION The study of physiological, receptor and metabolic lung parameters by PET is still in its infancy. No validated clinical applications of PET are available at present.

REFERENCES Ahluwalia B, Brownell GL, Hales C and Kazemi H (1981) Regional lung function evaluation with nitrogen13. Eur. J. Nucl. Med., 6, 453–457. Aigbirhio F, Pike VW, Francotte E, Jaeggi KA and Waters SL (1992) Simplified asymmetric synthesis and chiral analysis for S- [carbonyl-11C]CGP12177production. J.Label.Comp.Radiophop/iarm.,31,159-161. Barnes PJ (1993) Muscarinic receptor subtypes in airways. Life Sci., 52, 521–527. Barnes PJ (1997) Neural control of airway smooth muscle. The Lung, Crystal RG, West JB et al. (eds) 12691285. Raven Publishers: Philadelphia. Berridge MS, Cassidy EH, Terris AH and Vesselle J (1992) Preparation in in vivo binding of [11C]carazolol,

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a radiotracer for the beta-adrenergic receptor. Nucl. Med. Biol. 19, 563–569. Berridge MS, Nelson AD, Zheng LB, Leisure GP and Miraldi F (1994) Specific beta-adrenergic receptor binding of carazolol measured with PET. J.Nud.Med., 35, 1665–1676. Boullais C, Crouzel C and Syrota A (1986) Synthesis of 4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol- 2(11C)-one (CGP12177). J.Label.Comp.Radiopharm., 23, 565-567. Brady F, Luthra SK, Tochon-Danguy HJ, Steel CJ, Waters SL, Kensett MJ, Landais P, Shah F, Jaeggi KA and Drake A (1991) Asymmetric synthesis of a precursor for the automated radiosynthesis of S-(3 -tbutylamino-2'-hydroxypropoxy)-benzimidazol-2-[11C]one (S- [11C]CGP 12177) as a preferred radioligand for beta-adrenergic receptors, Int J Rad,Appl.lnstrum.[A], 42, 621 -628. Brodde OE (1991) Beta 1 - and beta 2-adrenoceptors in the human heart: Properties, function and alterations in chronic heart failure. Pharmacol.Rev.. 43, 203-242. Chan HK (1993) Use of single photon emission computed tomography in aerosol studies. J. Aerosol Med. 6, 23-36. Clark JC and Buckingham PD (1975) Short-Lived Radioactive Gases for Clinical Use. 171–214 Butterworths (London-Boston). Coates G, Fimau G, Meyer GJ and Gratz KF (1991) Noninvasive measurement of lung carbon-11 -serotonin extraction in man. J.Nucl.Med., 32, 729–732. Crouzel C, (1980) Anew radioisotope for lung ventilation studies: 19-neon. Eur.J.Nucl. 5, 431-434. Dolovich MB (2001) Measuring total and regional lung deposition using inhaled radiotracers. J. Aerosol Med. 14, 35–44. Doze P, Elsinga PH, de Vries EFJ, van Waarde A and Vaalburg W (2000) Mutagenic activity of a fluorinated analog of the beta-adrenoceptor ligand carazolol in the ames test. Nucl Med Biol. 27, 315-319. Elsinga PH, van Waarde A, Jaeggi KA, Schreiber G, Heldoorn M and Vaalburg W (1997) Synthesis and evaluation of (S)-4-(3-(2-[11C]isopropylamino)-2- hydroxypropoxy) -2H-benzimidazol -2-one ((S)[11C]CGP 12388) and (S)-4- (3-((l '-[18F]-fluoroisopropyl)amino)-2-hydroxypropoxy) -2H-berizirradazol-2-one ((S)-[18F]fluoro-CGP 12388) for visualization of beta-adrenoceptors with positron emission tomography, J. MedChem., 40, 3829–3835. Elsinga PH, van Waarde A, Doze P, Blanksma PK, Pieterman RM, Willemsen ATM and Vaalburg W (1999) Visualization of beta-adrenergic receptors in the human heart with (S)-[C-11]CGP 12388. J. Nucl. Med., 40, 87P. Even GA and Green MA (1989) Gallium-68-labeled macroaggregated human serum albumin,68Ga-MAA. Int.J.Rad.Appl.Instrum.B, 16, 319–321. Hakernarson H, Benard F, Khattri P, Shiue CY, Shiue GG, Karp JS, Grunstein MM and Alavi A (1997) An animal model for high resolution beta-adrenergic receptor imaging of asthma with S-1'-[F18]fluorocarazolol. J. Nucl Med. 38, 176P. Hayes MJ, Qing F, Rhodes CG, Rahman SU, Ind PW, Sriskandan S, Jones T and Hughes JM (1996) In vivo quantification of human pulmonary beta-adrenoceptors: effect of beta-agonist therapy. Am. J. Respir.Crit.Care Med, 154, 1277–1283. Hughes JM, Rhodes CG, Brudin LH, Valind SO and Pantin C (1987) Contribution of the positron camera to studies of regional lung structure and function. EurJ.Nucl.Med., 13 Suppl, S37–S41. Hwang DR, Eckelman WC, Mathias CJ, Petrillo EW, Jr., Lloyd J and Welch MJ (1991) Positron-labeled angiotensin-converting enzyme (ACE) inhibitor: fluorine-18-fluorocaptopril. Probing the ACE activity in vivo by positron emission tomography. J.Nucl.Med., 32, 1730-1737.

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Loch C, Maziere B and Comar D (1980) A new generator for ionic gallium-68. J.Nucl.Med., 21, 171–173. Lowe VJ and Sostraan HD (1998) Pulmonary embolism. In Nuc. Med. in Clin. Diag. and Treatment (eds Murray & Ell), Chrurchill Livingstone, Edinburgh, 37-54. Mak JC and Barnes PJ (1990) Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung. Am.Rev.Respir.Dis., 141, 1559–1568. Mak JC, Grandordy B and Barnes PJ (1994) High affinity [3H]formoterol binding sites in lung: characterization and autoradiographic mapping. EurJ.Pharmacol., 269, 35–41. McKenzie SA and Fitzpatrick ML (1981) Regional lung function in children: the accuracy of nitrogen-13 inhalation and infusion studies. Clin. Phys. Physiol. Meas. 2, 223–234. Murata K, Itoh H, Senda M, Todo G, Yonekura Y and Torizuka K (1986) Ventilation imaging with positron emission tomography and nitrogen-13. Radiology, 158, 303-307. Qing F, McCarthy TJ, Markham J and Schuster DP (2000) Pulmonary angiotensin-converting enzyme (ACE) binding and inhibition in humans. A positron emission tomography study. AmJ.Respir.Crit Care Med., 161, 2019–2025. Qing F, Rhodes CG, Hayes MJ, Krausz T, Fountain SW, Jones T and Hughes JM (1996) In vivo quantification of human pulmonary beta-adrenoceptor density using PET: comparison with in vitro radioligand binding. J. Nud. Med., 37, 1275–1281. Rhodes CG and Hughes JM (1995) Pulmonary studies using positron emission tomography. Eur.RespirJ., 8, 1001-1017. Roux FJ, Grandordy B and Douglas JS (1996) Functional and binding characteristics of long-acting beta 2agonists in lung and heart. AmJ.Respir.Crit Care Med., 153, 1489-1495. Schneeweiss A (1988) Cardiovascular drugs in children: angiotensin-converting enzyme inhibitors. Pediatr.CardioL, 9,109–115. Schuster DP (1998) The evaluation of lung function with PET. Semin.Nucl.Med., 28, 341–351. Senda M, Murata K, Itoh H, Yonekura Y and Torizuka K (1986) Quantitative evaluation of regional pulmonary ventilation using PET and nitrogen-13 gas. J. Nucl. Med. 27, 268–273. Syrota A, Pascal O, Crouzel M and Kellershohn C (1981) Pulmonary extraction of C-l 1 chlorpromazine, measured by residue detection in man. J.Nucl.Med., 22, 145–148. Tagil K, Evander E, Wollmer P, Palmer J and Jonson B (2000) Efficient lung scintigraphy. Clin.Physiol, 20, 95–100. Turton DR, Brady F, Pike VW, Selwyn AP, Shea MJ, Wilson RA and de Landsheere CM (1984) Preparation of human serum [methyl-11CJmethylalbumin microspheres and human serum [methyl11 C]methylalbumin for clinical use. IntJ.Appl.Radiat.Isot., 35, 337-344. Vaalburg W, Steenhoek A, Paans AMJ, Peset R, Reiffers S and Woldring MG (1981) Production of 13Nlabelled molecular nitrogen for pulmonary function studies. J. Lab. Compnds. Radiopharm., 18, 303–

308. Van der Mark TW, Rookmaker AEC, Kiers A, Peset R, Vaalburg W, Paans AMJ, Nickels RJ and Woldring MG (1984) Comparison of nitrogen-13 and xenon-133 for ventilation studies. J. Nucl. Med. 25, 1175-1182. Van der Wall H and Magnussen JS (1998) Non-embolic disease of the lungs. In Nuc. Med. Clin. Diag. and treatment, (eds Murray & Ell), Chrurchill Livinstone, Edinburgh, 55-66. van Waarde A, Elsinga PH, Doze P, Heldoorn M, Jaeggi KA and Vaalburg W (1998) A novel betaadrenoceptor ligand for positron emission tomography: evaluation in experimental animals. Eur. J. Pharmacol. 343, 289-2%.

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Visser TJ, van Waarde A, van der Mark TW, Kraan J, Elsinga PH, Pruim J, Ensing K, Jansen T, Willemsen AT, Franssen EJ, Visser GM, Paans AM and Vaalburg W (1997a) Characterization of pulmonary and myocardial beta-adrenoceptors with S-1 '-[fluorine- 18]fluorocarazolol. J. Nucl. Med,, 38,169–174. Visser TJ, van Waarde A, Jansen TJ, Visser GM, van der Mark TW, Kraan J, Ensing K and Vaalburg W (1997b) Stereoselective synthesis and biodistribution of potent [11CJ-labeled antagonists for positron emission tomography imaging of muscarinic receptors in the airways. JMed.Chem., 40, 11711124. Visser TJ, van Waarde A, Doze P, Elsinga PH, van der Mark TW, Kraan J, Ensing K and Vaalburg W (1998) Characterisation of beta2-adrenoceptors, using the agonist [llC]forrnoterol and positron emission tomography. EurJ PharmacoL, 361, 35–41. Visser TJ, van Waarde A, van der Mark TW, Kraan J, Ensing K, Willemsen AT, Elsinga PH and Vaalburg W (1999) Detection of muscarinic receptors in the human lung using PET. J. Nucl, Med., 40,1270–

1276. Wagner SJ and Welch MJ (1979) Gallium-68 labeling of albumin and albumin microspheres. J.Nucl.Med., 20, 428-433. Wollmer P, Pride NB, Rhodes CG, Sanders A, Pike VW, Palmer AJ, Silvester DJ and Liss RH (1982) Measurement of pulmonary erythromycin concentration in patients with lobar pneumonia by means of positron tomography. Lancet, 2, 1361–1364. Zheng LB, Berridge MS and Ernsberger P (1994)

Synthesis, binding properties and

18

F labeling of

fluorocarazolol, a high-affinity p-adrenergic receptor antagonist. J. Med.Chem., 37, 3219–3230.

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27. CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY Amin I. Kassis and S. James Adelstein Harvard Medical School Boston, Goldenson Building, 220 Longwood Avenue, Boston, MA 02115 INTRODUCTION For many years, the medical and the scientific communities have been intrigued by the possibility of using radionuclides in the treatment of cancer. Although early success was achieved with the use of radioiodine as iodide in the therapy of thyrotoxicosis and thyroid cancer, until recently the employment of unsealed radioactive sources for treatment has been largely unrealized. The problem has two components: first, the availability of radionuclides with appropriate physical properties and a chemistry that permits stable attachment to the targeting molecules; second, the development of targeting molecules with effective biologic properties. Within the past several years some of these difficulties have been overcome, and useful radiotherapeutic agents have been developed. This chapter, dedicated to Alfred P. Wolf's memory, summarizes the current state of radionuclide therapy. Among Al's several interests, this subject was one, and we are pleased to be asked to contribute to the book assembled in his honor. Our mutual interest in therapy was expressed some years ago when his laboratory at Brookhaven provided astatine-211 for some of our experiments. RADIOBIOLOGIC EFFECTS The deposition of energy by ionizing radiation is a random process. The energy absorbed by cells can induce certain molecular modifications that may lead to cell death. Despite the fact that this process is stochastic in nature, the death of a few cells, in general, within a tissue or an organ will not have a significant effect on function. As the dose increases, more cells will die with the eventual impairment of organ/tissue function. Molecular Lesions Damage to the genome is the basis for most radiation effects in cells. These radiobiologic effects display themselves generally as single- and double-strand breaks within DNA (SSB and DSB, respectively), base damage, and cross-links between DNA strands and nuclear proteins (Ward, 1986). Most cells, however, are equipped with a host of enzymes that are efficient at repairing damage to DNA. To aid in their function, most cells are also outfitted with machinery that holds up the mitotic cycle until repairs can be made. Repair of DNA damage from low-linear-energy-transfer (low-LET) radiations (photons and energetic electrons) is very efficient and sensitive to dose rate (Goodhead, 1985). On the other hand, the repair of damage from high-LET densely ionizing radiation (alpha particles and Auger effects in DNA) is more difficult.

Handbook of Radiopharmaceuticals. Edited by M. J. Welch and C. S. Redvanly. ©2003 John Wiley & Sons, Ltd

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Another manifestation of damage to DNA is the appearance of chromosomal and chromatidal aberrations. Chromosome-type aberrations are induced in Gt,-phase chromosomes or in unreplicated regions of S-phase chromosomes. Chromatid-type aberrations are induced in replicated regions of S-phase chromosomes and/or in G2-phase chromosomes. The shape of the dose—effect relation observed for chromosomal aberrations from low-LET radiations follows the general formula aD + p7)2. With increasing LET, the relation goes from linear—quadratic to linear. Lowering the dose rate also changes the low-LET aberration curve from the quadratic type to one increasingly more linear. Cellular Responses Survival. When cycling mammalian cells are irradiated by an external beam or the decay of a radiopharmaceutical, a certain fraction of the cells will lose their capacity to divide indefinitely, i.e., they fail to give rise to a clone of similar cells. Several in vitro assays have been described to measure the ability of cells to proliferate. In practice, these assays measure the capacity of cells to successfully reproduce and, thus, to form a colony. Using a colony-forming assay, it is possible to determine the decrease in survival, expressed as a surviving fraction, as a function of a graded radiation dose. Radiation survival curves are log-linear plots of surviving fraction (log) versus dose (linear). The shape of the survival curve constructed through such a set of survival points varies and will depend on certain biologic, physical, and chemical factors. In general, two types of dose-survival curves have been described (Figure 1). For the exponential survival curve, the slope is always constant and can be expressed by the following equation:

S/S0=&^

(1)

where S/SQ is the surviving fraction of the irradiated cells, D is the dose delivered, and DO is the dose needed to reduce survival to 0.37. This type of survival curve is observed when mammalian cells are exposed to high-LET radiation (e.g., alpha-particle emissions, DNA-incorporated Auger electron emitters).

S/S 0 = e

-(a D + p D2)

D o s e (D) Figure 1. Dose-Response survival curves for mammalian cells in vitro.

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769

In the second type of dose-response relationship, expressed by the sigmoidal survival curve (Figure 1), the efficiency of cell kill is not constant: at low doses, a slow decrease in survival is observed and the curve has a shoulder, at higher doses, an exponential decrease in survival is seen. The linear–quadratic equation for fitting this curve is:

_~ o0/0 / o 0— e Where a equals the rate of cell kill by a single-hit mechanism, D is the dose delivered, and P equals the rate of cell kill by a double-hit mechanism. This type of survival curve is routinely seen when mammalian cells are exposed to low-LET radiation (e.g., x-rays, beta particles, extranuclear Auger electrons). If radiation is protracted or the dose rate is low, as often occurs with radionuclides, the a term predominates. It is important to note that the a-to-p ratio is the dose at which cell killing by the linear and quadratic components is equal. Rewriting Equation 2 provides a method for calculating the rate constants a and P by graphic linearization (Chapman, 1980):

_—a_ When ln(S/So)/D is plotted against dose D, a linear relationship is obtained in which the y intercept with zero dose is the linear inactivation constant a and the slope of the line equals the quadratic inactivation constant p\ Such plots of cell inactivation can assist in making independent components of such inactivations readily apparent, and they have significantly improved the fit of data for mammalian cell survival curves and the resolution of problems associated with the conceptual difficulty of zero slope with zero dose. Whereas it is clear that radiopharmaceuticals labeled with radionuclides whose decay results in a purely exponential decrease in cell survival, with every decay leading to a corresponding decrease in survival (Equation 1), are preferable for radiotherapy, the exponential nature of both types of survival curve has important implications. In essence, it indicates that only the highest doses will reduce the number of viable cancer cells in a macroscopic tumor to fewer than one, and that no dose will be sufficiently large to eradicate with certainty 100% of the clonogenic cells, especially since the magnitude of the dose will always be limited by normal tissue tolerance. Nevertheless, the selection of appropriate carriers of radionuclides or a particular route of administration can lead to high target-to-normal-tissue ratios, reduce the dose to normal tissues, and result in effective doses to the targeted tissue. Division Delay. Dividing cells pass through four stages that are based on two observable events, mitosis (M) and DNA synthesis (S). The gap phases occurring before and after the S phase are known as G1 and Go, respectively. The delay in the progression of dividing cells through their cell cycle following irradiation is a well-known phenomenon. It is reversible and dose dependent, occurs only at specific points in the cell cycle, and is

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similar for both surviving and nonsurviving cells. Cells undergoing mitosis (M phase) continue through division basically undisturbed; those in the G, phase of the cell cycle have very little delay, those in S phase a moderate delay, while those in the premitotic G2 phase show maximum delay. The net result is that many cells accumulate at the G2/M boundary, and the ratio of mitotic cells to nonmitotic cells (i.e., the mitotic index) is altered. The length of the delay and the decrease in mitotic index are both functions of dose. Redistribution. The radiosensitivity of cells is a function of their position within the cell cycle. Cells in late S phase are most resistant (the survival curve has a wide shoulder), while those in the G2 and M phases are most radiosensitive (the survival curves have steep slopes and no shoulder). Consequently, following irradiation, the cells in the most sensitive phase will be killed differentially. This redistribution of cells will lead to partial cell-cycle synchrony and a change in the overall radiosensitivity of the cell cohort. One would expect a more radioresistant cell population, but in reality the population rapidly becomes desynchronized and the net effect is sensitization of the surviving population. Repair. Mammalian cells are generally capable of repairing some of the damage induced by radiation. This phenomenon is dose-rate dependent. As the dose rate decreases, the capacity of cells to repair radiation damage increases and this is manifested by a widening of the shoulder of the sigmoidal survival curve accompanied by an increase in D0 (e.g., X-ray irradiation). The shoulder is absent in survival curves that are purely exponential (e.g., alpha-particle irradiation). In essence, two types of repair have been described. The first, sublethal damage (SLD) repair, occurs when a dose of radiation is split into two fractions and sufficient time is allowed (0.5-2 h) for any/all of the radiation-induced damage to be repaired. Naturally, if no repair is allowed to occur, for example by the immediate application of a second dose of radiation, the cells will die. Sublethal damage and its repair have been shown to be important factors in the sparing of normal tissues during fractionated radiotherapy. The second, potentially lethal damage (PLD) repair, is observed only when mammalian cells are grown under suboptimal conditions following a single dose of radiation. Under such circumstances, an increase (rather than a decrease) in survival is observed. This phenomenon is believed to be a result of the delayed entry of the irradiated cells into mitosis, thereby allowing the cells to repair the PLD. Both SLD and PLD as well as their repair have been reported only for X-ray and gamma radiation (i.e., low-LET-type survival curves that have considerable shoulders) and are practically nonexistent for high-LET radiation (e.g., neutrons). Oxygen Effect.

Oxygen radiosensitizes mammalian cells to the damaging effects of radiation.

Consequently, hypoxic cells can be as much as threefold more radioresistant than well-oxygenated cells. It has been hypothesized that following irradiation, oxygen enhances free radical formation and/or blocks the reversal of certain reversible chemical alterations that have occurred. Here again, it is important to note that the oxygen effect is greatest for photons and high-energy beta particles (low-LET-type survival curves) and is practically absent for alpha particles and neutrons (high-LET-type survival curves). Tissue Responses The response of an organ or normal/cancerous tissue to ionizing radiation depends on the inherent sensitivity of the individual component cells and the kinetics of the cell population. The total radiation dose and the volume of tissue irradiated are also among the major determinants that regulate the nature of the

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77

response. However, factors such as a reduction of dose rate or fractionation of the total radiation dose can lead to cellular repair during irradiation and, therefore, mitigate the radiobiologic effects. Heterogeneity of Response. Following irradiation, the response of an organ/tissue/tumor will depend to a large degree on its inherent radiosensitivity. In the clinical situation, the term maximum tolerated dose (MTD) is used to indicate the highest dose that a normal organ can withstand. Once the MTD has been exceeded, the patient will exhibit the particular signs and symptoms associated with dysfunction of the organ being irradiated. The MTD to irradiation of assorted tissues and organs is variable. The tolerance doses for the gonads and bone marrow are low (100–200 cGy); those for the intestine, kidneys and heart are moderate (2000–4500 cGy); and those for mature bone and cartilage, the bladder and central nervous system are high (5000-7000 cGy).1 Consequently, knowledge of the MTD for the various organs and/or tissues being irradiated is quite important and can help to predict with some certainty the risks associated with a radiotherapeutic dose. Protraction and Fractionation Effects. Long before the in vitro techniques for tissue culture were developed, radiotherapists had realized that dividing the radiation dose into daily or weekly fractions is more effective in eradicating tumors than a single dose, while reducing the undesirable effects in normal tissues. The size and number of the fractions, the treatment time, and the total dose given depend mainly on the radiosensitivity of the tumor being eliminated and the tolerance of the surrounding normal tissues and organs. Since the response of a tumor to irradiation is determined in part by the depopulation of its dividing cells, it is expected that tumor cells react to dose fractionation in a fashion similar to that of acutely responding rather than late responding normal tissues, i.e., have high a/B ratios (higher ratios indicate that a lower proportion of the damage will be repaired). In fact, significant differences between the a/p ratios for tumors and normal tissues have been reported by Thames and Hendry (1987) and Williams et al. (1985). These authors have observed that while growing tumors exhibit a predominant o/p ratio of 10 to 20, normal tissues show a central tendency around 3 to 5. To the extent that such animal data are relevant to human tumors, it is not surprising that dose fractionation favors tumor control and minimizes normal tissue damage. Radiation Quality The goal of radiotherapy with internal emitters is to deliver therapeutic doses to a tumor without unduly affecting critical organs (e.g., the bone marrow). How effective the radiopharmaceutical will be is a function primarily of the absorbed-dose rate and the total absorbed dose delivered to the tumor and to normal tissues. The dose and its rate, in turn, depend on the injected activity, the kinetics of uptake and clearance of radioactivity within the tumor and normal tissues/cells, and the physical properties of the radionuclide (type of radiation, range of the emitted particles, etc.). Beta particle, alpha particle, and Auger electron emitters are three types of radionuclide that can be readily conjugated to various carrier molecules for tumor therapy (Table 1). Particles fron, these radioactive emissions produce tracks along which energy is transferred to and deposited in biologic matter. The intensity of the energy transfer varies and depends on the energy, charge, and mass of the traversing particle. The term linear energy transfer (LET) describes the transfer of energy (keV) along the track traversed (micrometer) by the particle. The LET of beta particle

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HANDBOOK OF RADIOPHARMACEUTICALS

emitters, such as iodine-131 and yttrium-90, is low and, depending on their energy, the particles traverse several millimeters. On the other hand, the LET of alpha particles emitted from astatine-211 and

Table 1. Physical Characteristics of Therapeutic Radionuclides Decay Mode

Particles

p

electrons

a

helium nuclei

EC/IC*

Auger electrons

Energy

medium-to-high (0.5-2.3 MeV) high (several MeV) very low (eV-keV)

Range

1-12 mm

50–100 urn several nm

*EC = electron capture; 1C = internal conversion.

bismuth-213 and of Auger electrons emitted by iodine-125 is high.2 While the tracks of alpha particles are several cell diameters in length, those of Auger cascade electrons are localized at the site of decay in a sphere of several nanometer radius. The tracks at low LET are sparsely ionizing and many (thousands) are needed to produce a detectable biologic effect (e.g., cell death). At high LET, the distance between ionizations becomes shorter and the tracks are dense so that these particles are much more efficient at producing lethal effects. For example, several alpha-particle traversals (fewer than five) through a cell nucleus or ~50 to 100 DNA-incorporated iodine-125 decays are sufficient to sterilize a cell. TARGETING PRINCIPLES IN RADIONUCLIDE THERAPY Choice of Radionuclide Although the choice of radionuclide cannot be easily separated from that of its targeting vector, certain general principles pertain to most applications. The physical attributes of interest are the particles emitted and the half-life. Beta Particles. Current radionuclide therapy in humans is based almost exclusively on beta-particleemitting isotopes. Although in theory both positively and negatively charged particles can be utilized, to date only negatron emitters have been employed. Typically, the electrons that are emitted from the nuclei of the decaying atoms (1 electron/decay) have various energies up to a maximum and thus have a distribution of ranges (Table 2). As each particle emitted traverses matter, it follows a contorted path, loses its kinetic energy, and eventually comes to a stop. Since the LET of these light, charged particles is very low (0.2 keV/pm), except for the few nanometers at the end of their range just before they stop, they are sparsely ionizing and quite inefficient at damaging DNA and killing cells. Consequently, their use as therapeutic agents predicates the presence of high radionuclide concentrations within the targeted tissue and the traversal of several thousand electrons per mammalian cell nucleus.

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

773

Table 2. Physical Characteristics of Beta Particle Emitters £p (max)

**P (max)

(keV)

(mm)

(keV)

(mm)

25.4 d

77

0.09

249

0.63

Er

9.4 d

99

0.14

350

1.1

Lu

6.7 d

133

0.23

497

1.8

Cu

61.9h

141

0.26

575

•2.1

8.0 d

182

0.39

610

2.3

Radionuclide

Half-Life

33p l69 l77 67

HI, I53

46.8 h

224

0.54

805

3.3

198

Au

64.8 h

312

0.89

961

4.2

109pd

13.5 h

361

1.1

1028

4.5

i86

3.8 d

349

1.1

1077

4.8

Sm

Re

165

Dy

2.3 h

440

1.5

1285

5.9

89

Sr

50.5 d

583

2.2

1491

7.0

32p

14.3d

695

2.8

1710

8.2

17.0h

764

3.1

2120

10.4

64.1 h

935

4.0

2284

11.3

188

Re

90y

*Mean (£j and maximum (E(maK}) energy of beta particles emitted per disintegration (Kocher, 1981). **Mean (R) and maximum (R( max )) beta-particle range in water [data from p. 206 in ICRU Report 37 (1984)].

An important ramification of the long range (mm) of each emitted electron is the production of cross-fire, a circumstance that negates the need to target every cell within the tumor and to internalize the radionuclide in each targeted cell. For microscopic disease, however, long-range and some medium-range emitters may deposit a significant fraction of the energy of their particles outside the tumor (Wheldon, 1994). This is particularly of concern in the selection of a radiopharmaceutical for the palliation of bone pain from metastatic osseous lesions (Bouchet et al, 2000). Long-range emitters, (e.g., phosphorus-32) run the risk of significantly irradiating bone marrow as well as bony lesions, whereas short-range emitters (e.g., phosphorus-33) are calculated to result in a significantly lower dose to bone marrow relative to bone/bony lesions. The matter of inhomogeneity in the distribution of a radionuclide and its consequent dose has also been addressed. O'Donoghue (1999) has derived a method for calculating an equivalent uniform biologically effective dose based on the absorbed-dose distribution represented by the biologically effective dosevolume histogram. For larger tumors he postulates that a combination of radionuclide and external beam therapy may result in optimal dose distribution.

774

HANDBOOK OF RADIOPHARMACEUTICALS

Many of the beta-particle-emitting radionuclides used for therapy also release gamma photons that generally do not add significantly to the dose delivered to the target tissue. However, these photons may contribute considerably to the whole-body dose. For example, 100 mCi of iodine-131 distributed throughout the whole body would deposit about 60 cGy per day. Since the bone marrow is usually the dose-limiting organ (200–300 cGy), the success or failure of therapy will depend on not exceeding this MTD. Alpha Particles. An alpha particle that is emitted by any of the radionuclides suitable/available for therapy (Table 3) is identical to a helium nucleus with energies ranging from 4 to 9 MeV. The corresponding tissue ranges are 40 to 100 pm. Alpha particles travel in straight lines, initially depositing their energy, in soft tissue, at approximately 80 keV/pm and increasing their rate of energy deposition toward the end of their tracks, i.e., in their Bragg peak region. The typical energy deposition from an alpha particle traversing the diameter of a cell nucleus is about 600 keV; this translates to an absorbed dose of about 0.14 Gy to the cell nucleus per hit. These numbers depend, among other parameters, on the size of the cell nucleus. Whereas the stochastics of energy distribution are unimportant for absorbed doses greater than 1 Gy for beta particles, because each cell experiences thousands of individual particle tracks, the average specific energy deposited per unit mass (i.e., the absorbed dose) is not a suitable parameter for cells traversed by a few alpha particles. Several authors have thus described microdosimetric approaches for calculating the specific energy deposited in individual cells and predicting the response of cells to alpha-particle irradiation (Charlton & Sephton, 1991; Charlton et al., 1994); others (for example, Roeske & Stinchcomb, 1999) have related the specific energy distributions to the fraction of cell survivors. The magnitude of cross-dose (from radioactive sources associated with one cell to an adjacent cell) is an important factor when evaluating alpha particles for therapy. This will vary considerably depending on the size of the labeled cell cluster and the fraction of cells labeled (Goddu et al., 1994).

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

775

Table 3. Physical Characteristics of Alpha Particle Emitters

Radionuclide

21

'At

Half-Life

E;

R"

(MeV)

(Mm)

7.2 h

6.79

60

212

Bi

60.6 min

7.80

75

213

Bi

45.7 min

8.32

84

*Mean energy (E a) of alpha particles emitted per disintegration (Kocher, 1981). **Mean range (R a) of alpha particles calculated using second order polynomial regression fit [data from p. 256 in ICRU Report 49 (1993)]: R = 3.87£ + 0.75E2 - 0.45, where R is the range (u,m) in unit density matter and E is the alpha-particle energy (MeV).

Finally, when the alpha particle emitter is covalently bound to nuclear DNA (i.e., in the case of cell selfirradiation), heavy ion recoil of the daughter atom must also be considered (Walicka et al., 1998c). Auger Electrons. During the decay of certain radioactive atoms, a vacancy is formed (most commonly in the K shell) as a consequence of electron capture (EC), with the prompt emission of a neutrino from the atomic nucleus and/or internal conversion (1C). The vacancy is rapidly filled by an electron dropping in from a higher shell. As a result of nuclear rearrangements, some Auger electron emitters also emit a gamma photon that may itself be converted to a nuclear vacancy resulting in a second shower of Auger electrons (e.g., iodine-125). Similarly, other metastable nuclei (e.g., technetium-99m, bromine-80m) emit a gamma photon which may or may not be converted into an electron vacancy (only 3% of the 140 keV gamma photons of technetium-99m are converted). This process (creation of an electron vacancy within a shell and its filling up) leads to a cascade of atomic electron transitions that move the vacancy towards the outermost shell. Each inner-shell electron transition results in the emission of either a characteristic x-ray photon or an Auger, Coster-Kronig, or super Coster-Kronig electron (collectively called Auger electrons). These electrons are monoenergetic. Typically an atom undergoing EC and/or 1C emits several Auger electrons with energies ranging from a few eV to approximately 80 keV. Thus, the range of Auger electrons in water is from a fraction of a nanometer to several tens of microns. In some cases (e.g., iodine-125, platinum195m) as many as 20 to 33 Auger electrons are emitted per decay on average (Table 4). In addition to the shower of low-energy electrons, this form of decay leaves the daughter atom with a high positive charge resulting in subsequent charge transfer processes.

776

HANDBOOK OF RADIOPHARMACEUTICALS Table 4. Physical Characteristics of Auger Electron Emitters Radionuclide

Half-Life

Electrons per Decay*

Average Energy (eV) Deposited in 5-nm Sphere

Cr

27.7 d

6

210

Ga

3.3d

5

260

75

120 d

7

270

"-Tc

6h

4

280

77

57 h

7

300

I

13.1 h

11

420

'In

2.8 d

8

450

.25,

60.1 d

20

1000

201 r^l

51 67

Se Br

123 11

73.1 h

20

1400

193mpt

4.3d

26

1800

.95mpt

4.0 d

33

2000

'Average yield of Auger and Coster-Kronig electrons.

Unlike the long range of alpha and beta particles discussed above, which is greater than that of the diameter of mammalian cells (10-15 pm), the short range of Auger electrons requires their close proximity to radiosensitive targets for radiotherapeutic effectiveness. This is essentially a consequence of the precipitous drop in energy density as a function of distance in nanometers [for example, Figure 1 in Kassis et al., (1982) and Figure 4 in Kassis et al., (1987a)]. These Auger electron emitters need to be located in the cell nucleus, close to or incorporated into DNA. In summary, the type of decay and decay particle determine the range of the latter and the ionization density it produces. Generally, beta particles travel millimeters, alpha particles, micrometers, and Auger electrons, nanometers (Table 1). Thus, each of these is increasingly fastidious with regard to its proximity to biologic targets. Half-Life. Because many biologic responses to radiation are sensitive to dose rate as well as total dose, the physical half-life of the radionuclide employed and the tumor/normal tissue residence half-life can be of consequence. For a radiopharmaceutical with an infinite residence time in a tumor or tissue, a radionuclide with a long physical half-life will deliver more decays than one with a short half-life if both have the same initial radioactivity. Moreover, there can be a striking difference in the time-dependent dose rate delivered by the two. If the number of radionuclide atoms per unit of tissue mass is n and the energy emitted (and absorbed) per decay is E, then the absorbed-dose rate is proportional to nE/T where T is the half-life. The ratio EIT is thus one important indicator of the intrinsic radiotherapeutic potency of the radionuclide (O'Donoghue, 1994). In general, for biologic reasons, higher dose rates delivered over shorter treatment times are more effective than lower dose rates delivered over longer periods. Thus, a radionuclide with a

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

777

shorter half-life will tend to be more biologically effective than one with a similar emission energy but longer half-life. In addressing this phenomenon, ODonoghue & Wheldon (1996) have noted that when the same number of iodine-125 and iodine-123 atoms are bound to a tumor, their relative effectiveness depends on the tumor doubling time and the rate at which the radiopharmaceutical dissociates from the target. When both are very long, the longer-lived iodine-125 is theoretically more effective; otherwise the shorter-lived iodine-123 is preferred. Choice of Vector or Ligand Of equal importance to the choice of radionuclide in cancer therapy is that of the chemical carrier. For it is the properties of the latter upon which targeted therapy depends. The selection of a suitable carrier molecule rests on many factors. These include (i) biologic specificity and in vivo stability, (ii) the biologic mechanism(s) that bind it to target cells and the affinity of the carrier for these sites, and (iii) the stability of the complex thus formed. Obviously, the chemical properties of the carrier molecule must enable the conjugation of a therapeutic radionuclide without degradation of the intrinsic characteristics of the molecule. Finally, the physical half-life of the radionuclide must be at least equal to, and preferably much longer than, the biologic half-life of the carrier molecule. One of the simplest targeting agents is radioiodine, administered as the iodide for treating functional cancer of the thyroid and potentially breast cancers that display the iodine transporter. In the palliation of metastatic bone pain, a simple cation, radiostrontium, has been used. Its targeting relies on the accretion of alkaline earth cation congeners of calcium to bony surfaces. The oxides of radiophosphorus as orthophosphates and the somewhat more complex phosphonates labeled with radiorhenium and radiosamarium have also been proposed. Short peptides labeled with yttrium-90 have been used to target neuroendocrine tumors (Smith et al., 2000). Meta-[131I]iodobenzylguanidine (M131IBG), an iodinated neurotransmitter, is taken up by neural crest tumors (Loh et al., 1997). Radioyttrium and rare earth (e.g., dysprosium) colloids have been employed in the treatment of advanced arthritis. The colloids, injected into the joint cavity, are taken up by synovial macrophages (Shortkroff et al., 1993). Radiocolloid labeled with the alpha particle emitter astatine-211 has been successfully employed in treating ovarian ascites tumors in mice (Bloomer et al,, 1981, 1984). Radioiodinated pyrimidine deoxyribose nucleosides (e.g., radioiododeoxyuridine) have been used for the experimental treatment of ovarian, brain, and spinal cord tumors in animals (Bloomer & Adelstein, 1977; Baranowska-Kortylewicz et al., 1991; Kassis et al, 1998, 2000c). Because, the carrier is incorporated into the DNA of dividing cells, it is most effective when labeled with Auger-electron-emitting radionuclides. Successful therapy of lymphomas and leukemias has been achieved with intact antibodies and antibody fragments labeled with alpha- and beta-particle-emitting radionuclides (Foon, 2000). Specificity in this case relies on tumor-associated antigens located on the malignant cell surface.

778

HANDBOOK OF RADIOPHARMACEUTICALS

Properties of Targets Important factors in the case of tumors are their accessibility, the number of binding sites, and the distribution of binding sites among the targeted and nontargeted cells and their relationship to the cell cycle. The microscopic environment of the target, including tumor vascularity, vascular permeability, oxygenation, as well as microscopic organization and architecture, is also extremely important (Jain, 1990; Netti et al., 1999). To optimally target a radiopharmaceutical, the route of administration (e.g., intravenous, intralymphatic, intraperitoneal, intracerebral, intravesical, intrathecal, and intrasynovial) must also be considered. Some pathways may provide a mechanical means for maximizing tumor-to-nontumor ratios. Lastly, specific activity of the radiopharmaceutical should be taken into account, especially when receptors can easily be saturated and weaker nonspecific binding competes with the target. On the other hand, certain treatments, including the use of radiolabeled antibodies, are assisted by a mass effect and optimized by the addition of unlabeled immunoglobulin. EXPERIMENTAL THERAPEUTICS Beta Particle Emitters The toxicity of beta-particle-emitting radionuclides has been assessed in vitro for many years. Early work, carried out by radiobiologists, concentrated on tritium and carbon-14, whereas later studies examined the therapeutic potential of more energetic beta particle emitters (Table 2). In these studies, the survival curves either have a distinct shoulder (Burki et al., 1973; Chan et al., 1976; Govindan et al., 2000) or are of the high-LET type (Ragni & Szybalski, 1962; Burki et al., 1973; Liber et al., 1983). Invariably, the D0 calculated is several thousand decays (Figure 2). Similarly, the decay of such radionuclides (e.g., tritium, carbon-14, and iodine-131) has been shown to lead to various molecular alterations (e.g., SSB, DSB, chromosomal aberrations), but only when the cells are exposed to very large numbers of decays (10 000150 000) (Cleaver & Burki, 1974; Chan et al., 1976; Gutierrez et al., 1998; Hengstler et al., 2000).

779

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

125,

I-DR (cytoplasm)

1E-3

'lUdR (DMA-bound) (DMA-bound) 2,500

5,000

7,500

10,000

Decays per Cell

Figure 2. Survival of mammalian cells following exposure to

I25,

I decays [DNA-incorporated (Kassis et al.,

I987a) or within the cytoplasm (Kassis et al., 1987b)]. The survival curve for 13l IUdR is included for comparison (Chan et al., 1976). Despite the rather low in vitro toxicity of beta-particle-emitting radionuclides, they continue to be pursued for targeted therapy. This is in part due to their availability and the long range of the emitted electrons, which can lead to the irradiation of all the cells that are within the maximum range and path of the particle. As mentioned above, the main advantage of cross-fire is that it negates the necessity of the radiotherapeutic agent being present within each of the targeted cells, i.e., it permits a certain degree of heterogeneity. However, three factors will determine whether an effective therapeutic dose will be delivered to the targeted tissue. First, it is essential that the targeted therapeutic agent concentrate within foci throughout the targeted tissue. Second, the distances between these hot foci must be equal to or less than twice the maximum range of the emitted energetic beta particles. Third, the concentration of the radiotherapeutic agent within each hot focus must be sufficiently high to produce a cumulative cross-fire dose to the surrounding targeted cells of ~10 000 rad. Since dose is inversely proportional to the square of the distance, the concentration of the therapeutic agent needed to deposit such cytocidal doses will decrease precipitously when the distance between the hot foci decreases. Experimentally, these predictions have been substantiated in various animal-tumor therapy studies. In most of these studies, radionuclides have been targeted after conjugation to monoclonal antibodies. For example, investigators have assessed the therapeutic efficacy of 131I-labeled monoclonal antibodies in rodents bearing subcutaneous tumors. Although a substantial proportion of cells within a tumor mass show reduced/no expression of the targeted antigen and, therefore, are not targeted by the radioiodinated antibody,

131

I-

labeled antibodies that localize in high concentrations in tumors are therapeutically efficacious and can lead to total tumor eradication in some instances (Esteban et al., 1987; Riklund et al, 1990; Buchsbaum et al.,

HANDBOOK OF RADIOPHARMACEUTICALS

780

1995; Sato et al., 1999). Thus, even when iodine-131 is not-so-uniformly distributed within a tumor, the decay of this radionuclide can lead to tumor sterilization as long as it is present in sufficiently high concentrations (Figure 3). Similar results have also been reported with other beta-particle-emitting isotopes, in particular yttrium-90 (Otte et al., 1998; Chinn et al., 1999; Govindan et al., 2000; Watanabe et al., 2000) and copper-67 (DeNardo et al., 1997a, 1997b).

10

15

20

25

30

35

40

Time (days) Figure 3. Tumor growth in mice post treatment with 1995).

131

I-labeled monoclonal antibody (Buchsbaum et al.,

Several "two- and three-step" approaches have also been proposed to target radioactivity to a tumor (Hnatowich et al., 1987; Goodwin et al., 1988; Le Doussal et al., 1990; Kassis et al., 1996; Axworthy et al., 2000). In general, an antibody that is not internalized by the targeted cell is injected prior to the administration of a small radiolabeled molecule that has a strong affinity to the antibody. The most extensively studied approach utilizes the high avidity of avidin (Av) or streptavidin (SA) for biotin, a vitamin found in low concentration in blood and tissues. Because the noncovalently bound Av/SAv-biotin complex has an extremely low dissociation constant (k^) of about 10–15 M, investigators have used these two molecules as binding pairs to bridge molecules that have no affinities for each other and to target various radionuclides. A recent report (Axworthy et al., 2000), which examined the efficacy of this approach in tumor-bearing mice, has demonstrated that 100% of the mice treated with the 90Y-labeled biotin derivative have been cured of their disease (Figure 4).

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

o

781

10 1-

**

"J3 S c

Untreated 800 M.CI »»Y-DOTA-B (alone) 10/10 cures

0.10.01

I

0

I

I

I

I

10 20 30 40 50 Days post-treatment

Figure 4. Therapeutic activity of ^Y-DOTA-biotin post monoclonal-antibody-streptavidin conjugate administration in tumor-bearing mice (Axworthy et al., 2000). • and •: controls; A: 800 uCi ^ following pretargeting.

Alpha Particle Emitters The potential application of alpha-particle-emitting radionuclides as targeted therapeutic agents has been of interest for more than 25 years. If selectively accumulated in the targeted tissues (e.g., tumors), the decay of such radionuclides with their high LET and short range of a few (~5-10) cell diameters should result in highly localized energy deposition in the targeted tumor cells and minimal irradiation of surrounding normal host tissues. As a consequence of the LET of these particles (80 keV/um), a single traversal of an alpha particle through a mammalian cell nucleus will kill a cell (Kassis et al., 1986; Raju et al., 1991; Charlton et al., 1994; Walicka et al., 1998c). In comparison, the LET of negatrons emitted by the decay of beta emitters is very much smaller (0.2 keV/pm) and thus ~20,000 beta particles must traverse a cell nucleus for its sterilization. The investigation of the therapeutic potential of alpha particle emitters has focused mainly on three radionuclides: astatine-211 (21lAt), bismuth-212 (212Bi), and bismuth-213 (213Bi) (Table 3). In vitro studies (Kassis et al., 1986; Charlton et al., 1994) have demonstrated that the decrease in survival of mammalian cells after exposure to uniformly distributed alpha particles from these radionuclides is monoexponential (Figure 5) but that, as predicted theoretically (Humm et al., 1990) and experimentally (Walicka et al., 1998c), these curves develop a tail when the dose is nonuniform (Figure 5). Such studies have shown that (i) these alpha particle emitters are highly toxic (Kassis et al., 1986; Kurtzman et al., 1988; Charlton et al., 1994; Larsen et al., 1994, 1998; Strickland et al., 1994; Palm et al., 1998; Nikula et al, 1999), (ii) cells in

HANDBOOK OF RADIOPHARMACEUTICALS

782

monolayer require more alpha-particle traversals than spherical cells (Kassis et al., 1986; Charlton et al., 1994), and (iii) only a few (~l-4) alpha-particle traversals through a mammalian cell nucleus are necessary to sterilize a cell (Kassis et al., 1986; Charlton etal., 1994; Nikula et al., 1999).

1

Theoretical Expectations

v

nonuniform " v distribution

uniform* dtotributloiA

Dose

•* 211

AtUdR-DOa=1.3

a =7.1

Bi (Bi-DtPA) - DQ=3.0 At (astatide) - D =0.9

0

10

20

Alpha-Particle Traversals

Figure 5. Survival of mammalian cells exposed in suspension to 2llAt-astatide (Kassis et al., 1986),212 BiDTPA (Charlton et al, 1994), or 211 AtUdR (only 50% of cells labeled) (Walicka et al., 1998c). Insert theoretical expectations (Humm et al., 1990). At the molecular level, the traversal of alpha particles through a mammalian cell nucleus leads to the efficient production of chromosomal aberrations and DSB. For example, the incubation of mammalian cells with 211At-astatide causes a significant increase in chomosomal aberrations (Kassis et al., 1986), but these decline with the passage of time (Figure 6). More recently, Walicka et al. (1998c) have established that more than 10 DSB are produced per decay of DNA-incorporated astatine-211, a value much higher than that obtained following the decay of the DNA-incorporated Auger electron emitter iodine-125 (Walicka et al., 1998a; Kassis et al., 2000a).

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

Figure 6. DNA damage post exposure to

783

At (Kassis et al., 1986).

Investigators have also assessed the therapeutic potential of alpha particle emitters in tumor-bearing animals (Bloomer et al., 1981, 1984; Kozak et al., 1986; Harrison & Royle, 1987; Macklis et al., 1988; Link & Carpenter, 1990; Zalutsky et al., 1994; Kennel & Mirzadeh, 1998; Larsen & Bruland, 1998; Adams et al., 2000). Bloomer et al., (1981, 1984) reported a dose-related prolongation in median survival when mice bearing an intraperitoneal murine ovarian tumor are treated with 2llAt-tellurium colloid administered directly into the peritoneal cavity. Moreover, this alpha-particle-emitting radiocolloid is curative without serious morbidity, whereas colloids of beta-particle-emitting radionuclides (phosphorus-32, yttrium-90, dysprosium-165) are not (Figure 7). In another set of in vivo studies examining the therapeutic efficacy of a 2l2 Bi-labeled monoclonal antibody, the radionuclide is most effective when used with a carrier having target specificity (Mackiis et al., 1988). The preliminary results of clinical trials in which patients received 211 At/2l3Bi-labeled antibodies have been reported only in abstracts (Jurcic et al., 1997; Sgouros et al., 1997; Ballartgrud et al., 1998; Akabani et al., 2000), but they seem to be cautiously promising. Auger Electron Emitters The toxicity to mammalian cells of radionuclides that decay by EC and/or IC has, for the most part, been established with the Auger-electron-emitting radionuclide iodine-125. Because of its predominant (93%) IC decay following EC, iodine-125 is a prolific emitter of Auger electrons [mean of ~20 per decaying atom (Charlton & Booz, 1981; Charlton, 1986; Charlton et al., 1987; Pomplun et al., 1987)]. Dosimetric calculations have predicted that the electrons most frequently produced by iodine-125 dissipate their energy in the immediate vicinity of the decaying atom and deposit 108 to 109 rad/decay within two-nanometer spheres around the decay site (Sastry & Rao, 1984; Kassis et al., 1985, 1987a). Thus, the biologic effects are expected to depend critically on the proximity of the radionuclide to DNA.

784

HANDBOOK OF RADIOPHARMACEUTICALS

Figure 7. Percentage change in median survival of tumor-bearing mice treated with a- and p-emitting radiocolloids (Bloomer et al., 1984).

The radiotoxicity of iodine-125 has been studied in vitro under a number of conditions: (i) when the radioelement is incorporated into the DNA duplex as iododeoxyuridine (lUdR) (Hofer & Hughes, 1971; Porteous, 1971; Burki et al., 1973; Bradley et al., 1975; Hofer et al., 1975; Chan et al., 1976; Kassis et al., 1987a); (ii) when it is intercalated between the stacked bases of DNA as 3-acetamido-5-[l25I]iodoproflavine (AI25IP) (Kassis et al., 1988, 1989); (iii) when it is bound to the minor and major grooves of DNA as an iodinated Hoechst dye (Walicka et al., 1999); (iv) as a radiolabeled triplex-forming oligonucleotide (Sedelnikova et al., 1998); (v) when it is bound to transcription elements as iodinated tamoxifen and estrogen (Bloomer et al., 1983; Yasui et al., 2001); and (vi) when it is located in the cytoplasm of cultured cells as iodorhodamine (I25I-DR) (Kassis et al., 1987b). The decay of iodine-125 incorporated into cellular DNA as RJdR leads to an exponential decrease in clonal survival (Figure 2). Similar responses are seen with bromine-77 (Kassis et al., 1982) and iodine-123 (Makrigiorgos et al., 1989). When plotted as a function of the total number of decays, the three curves have different slopes. If the dose to the cell nucleus is calculated, a single cell survival curve is obtained (Figure 8), suggesting that the radiation dose to the cell nucleus is a parameter that describes these observed biologic effects adequately. When the intercalating agent A125IP is incubated with cells, the survival curve is still exponential, but each decay is 1.5 times less effective than 125IUdR, i.e., its relative biologic effectiveness (RBE) is lower (Figure 8). On the other hand, a recent study of survival on exposure to an I25 l-labeled estrogen ligand reports a D0 similar to 125IUdR (Yasui et al., 2001).

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

785

I-DR RBE=1.3 1E-3

0

250

500

750

1000

Radiation Dose to Cell Nucleus (cGy)

Figure 8. Survival fraction (S750) of V79 cells plotted as function of radiation dose to cell nucleus. RBE of each agent is calculated at the D37 (Kassis et al., 1988).

Contrary to these survival curves, incubation of mammalian cells with an 125I-labeled DNA groove binder (Hoechst 33342) results in a log-linear-quadratic survival curve (Walicka et al., 1999). A similar lowLET-like survival curve is also seen when iodine-125 decays within the cytoplasm (Figure 8). Furthermore, the RBE of these agents is even lower, i.e., many more decays are required for equivalent toxicity (Kassis et al.. 1987b). From these experiments, it must be concluded that the position of the Auger electron emitter with regard to DNA is a major determinant of radiotoxicity and that under certain circumstances decays result in a highLET-like response. The amount of energy deposited in the nucleus is also important, and decays taking place in the cytoplasm are much less effective. Molecular studies have shown that the decay of iodine-125 in iodouracil leads to the creation of carbon fragments from the pyrimidine ring (Halpern & Stocklin, 1977a, 1977b). The incorporation of the radionuclide into duplex oligonucleotides leads to shattering of the nucleic acid strands (Martin & Haseltine, 1981). Similarly, its binding to the minor groove of plasmids results in a high yield of DSB (Kassis et al., 1999a, 1999b). Whereas such studies have predicted correctly that the decay of DNAincorporated iodine-125 would be highly cytotoxic to mammalian cells, they have assumed that the cytotoxicity of 125IUdR would be due solely to the deposition of energy directly into the DNA molecule. Experiments with radical scavenging agents have established that this is not the case and that clusters of watery radicals, formed following the decay of iodine-125 within chromatin, damage not only DNA at the site of decay but also a number of distant bases due to the packaging and compaction of DNA (Walicka et al., 1998a; Kassis et al., 2000a). As a result, the cyotoxicity of 125IUdR in mammalian cells can be modified strikingly by radical scavengers (Walicka et al., 1998b; Bishayee et al., 2000; Walicka et al., 2000). Indeed, it now seems likely that indirect effects caused by clusters of aqueous radicals are the principal mechanism of cell damage.

786

HANDBOOK OF RADIOPHARMACEUTICALS

The extreme degree of cytotoxicity observed with DNA-incorporated iodine-125 has been exploited in experimental radionuclide therapy using 125IUdR. Since this radiopharmaceutical breaks down rapidly after systemic administration, all of the studies have been carried out in animal models and in patients where locoregional administration is feasible. For example, the injection of 125IUdR into mice bearing an intraperitoneal ascites ovarian cancer has led to a 5-log reduction in tumor cell survival (Bloomer & Adelstein, 1977). Similar results are obtained with 123IUdR (Baranowska-Kortylewicz et al., 1991). When 125 IUdR is administered intracerebrally to rats bearing an intraparenchymal gliosarcoma, the survival of treated animals is significantly prolonged (Kassis et al., 1990, 1998). Therapeutic doses of125IUdR injected intrathecally into rats with 9L rat gliosarcoma or human TE-671 rhabdomyosarcoma intrathecal tumors significantly delay the onset of paralysis in these animals (Sahu et al., 1997; Kassis et al., 2000b). Most recently, Kassis et al. (2000c) have shown that the therapeutic efficacy of l25IUdR is substantially enhanced by the co-administration of methotrexate, an antimetabolite that enhances lUdR uptake by DNAsynthesizing cells (Figure 9). This chemo-radio combination in fact leads to 5–6 log kill and cures -30% of the animals.

MTX, sIUdR, MTX 125 IUdR: M=82 d

0

25

50

75

100

125

Days after Tumor Cell Injection Figure 9. Therapy with intrathecal

l25

IudR ± methotrexate in rats bearing human intrathecal rhabdomyosarcoma

(Kassis et al., 2000c).

SUMMARY A significant increase in our understanding of the dosimetry and therapeutic potential of various modes of radioactive decay has heightened the possibility of utilizing specifically labeled carriers in cancer therapy. Moreover, as a consequence of the great strides in genomics, the development of more precise targeting molecules is at hand. Further progress in the field of targeted radionuclide therapy will be made by the judicious design of radiolabeled molecules that match the physical and chemical characteristics of both the radionuclide and the carrier molecule with the clinical character of the tumor.

CONSIDERATIONS IN THE SELECTION OF RADIONUCLIDES FOR CANCER THERAPY

78'

Footnotes 1 These doses are for external beam therapy. With radionuclides, where the dose rate is much lower, the MTD may be somewhat higher. 2 Actually, for Auger emitters, the electrons produce ionizations that are clustered around the point of decay, i.e., they are not along a linear track.

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At-labeled monoclonal antibody (MAB) using histological images of malignant brain tumors. J.

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I-labeled monoclonal antibody

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Kassis Al, Fayad F, Kinsey BM, Sastry KSR and Adelstein SJ (1989) Radiotoxicity of an I25l-labeled DNA intercalator in mammalian cells. Radiat. Res., 118, 283–294. Kassis Al, Van den Abbeele AD, Wen PYC, Baranowska-Kortylewicz J, Aaronson RA, DeSisto WC, Lampson LA, Black PM and Adelstein SJ (1990) Specific uptake of the Auger electron-emitting thymidine analogue 5-[123I/125I]iodo-2'-deoxyuridine in rat brain tumors: diagnostic and therapeutic implications in humans. Cancer Res., 50, 5199-5203. Kassis Al, Jones PL, Matalka KZ and Adelstein SJ (1996) Antibody-dependent signal amplification in tumor xenografts following pretreatment with biotinylated monoclonal antibody and avidin or streptavidin. J. Nucl. Med., 37, 343–352. Kassis Al, Wen PY, Van den Abbeele AD, Baranowska-Kortylewicz J, Makrigiorgos GM, Metz KR, Matalka KZ, Cook CU, Sahu SK, Black PM and Adelstein SJ (1998) 5-[125I]iodo-2'-deoxyuridine in the radiotherapy of brain tumors in rats. J. Nucl. Med., 39, 1148–1154. Kassis Al, Harapanhalli RS and Adelstein SJ (1999a) Comparison of strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125. Radiat. Res., 151, 167–176. Kassis Al, Harapanhalli RS and Adelstein SJ (1999b) Strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125: direct compared to indirect effects. Radiat. Res., 152, 530–538. Kassis Al, Walicka MA and Adelstein SJ (2000a) Double-strand break yield following I25I decay: effects of DNA conformation. Acta Oncol., 39, 721–726. Kassis Al, Dahman BA and Adelstein SJ (2000b) In vivo therapy of neoplastic meningitis with methotrexate and 5-[125I]iodo-2 '-deoxyuridine. Acta Oncol., 39, 731–737. Kassis Al, Dahman BA and Adelstein SJ (2000c) Therapy of neoplastic meningitis with methotrexate and 5-[125I]iodo-2'-deoxyuridine. J. Nucl. Med., 41(5, suppl), 275P. Kennel SJ and Mirzadeh S (1998) Vascular targeted radioimmunotherapy with 213Bi—an a-particle emitter. Nucl. Med. BioL, 25, 241–246. Kocher DC (1981) Radioactive Decay Data Tables: A Handbook of Decay Data for Application to Radiation Dosimetry and Radiological Assessments. National Technical Information Center, US Department of Energy: Springfield, Virginia. Kozak RW, Atcher RW, Gansow OA, Friedman AM, Hines JJ and Waldmann TA (1986) Bismuth-212labeled anti-Tac monoclonal antibody: a-particle-emitting radionuclides as modalities for radioimmunotherapy. Proc. Natl. Acad. Sci. USA, 83,474–478. Kurtzman SH, Russo A, Mitchell JB, DeGraff W, Sindelar WF, Brechbiel MW, Gansow OA, Friedman AM, Hines JJ, Gamson J and Atcher RW (1988) 2l2 Bismuth linked to an antipancreatic carcinoma antibody: model for alpha-particle-emitter radioimmunotherapy. J. Natl. Cancer Inst., 80, 449–452. Larsen RH and Bruland 0S (1998) Intratumour injection of immunoglobulins labelled with the a-particle emitter 211At: analyses of tumour retention, microdistribution and growth delay. Br. J. Cancer, 77, 1115-1122. Larsen RH, Bruland 0S, Hoff P, Alstad J, Lindmo T and Rofstad EK (1994) Inactivation of human osteosarcoma cells in vitro by 2ll At-TP-3 monoclonal antibody: comparison with astatine-211-labeled bovine serum albumin, free astatine-211 and external-beam X rays. Radiat. Res., 139, 178–184. Larsen RH, Akabani G, Welsh P and Zalutsky MR (1998) The cytotoxicity and microdosimetry of astatine211-labeled chimeric monoclonal antibodies in human glioma and melanoma cells in vitro. Radiat. Res., 149, 155–162.

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Le Doussal J-M, Gruaz-Guyon A, Martin M, Gautherot E, Delaage M and Barbel J (1990) Targeting of indium 111-labeled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: imaging of tumors hosted in nude mice. Cancer Res., 50, 3445–3452. Liber HL, LeMotte PK and Little JB (1983) Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DMA of human lymphoblast cells. Mutat. Res., 1ll, 387-404. Link EM and Carpenter RN (1990) 211At-methylene blue for targeted radiotherapy of human melanoma xenografts: treatment of micrometastases. Cancer Res., 50, 2963–2967. Loh K-C, Fitzgerald PA, Matthay KK, Yeo PPB and Price DC (1997) The treatment of malignant pheochromocytoma with iodine-131 metaiodobenzylguanidine (131I-MIBG): a comprehensive review of 116 reported patients. J. Endocrinol. Invest., 20, 648–658. Macklis RM, Kinsey BM, Kassis AI, Ferrara JLM, Atcher RW, Hines JJ, Coleman CN, Adelstein SJ and Burakoff SJ (1988) Radioimmunotherapy with alpha-particle-emitting immunoconjugates. Science, 240, 1024–1026. Makrigiorgos GM, Kassis Al, Baranowska-Kortylewicz J, McElvany KD, Welch MJ, Sastry KSR and Adelstein SJ (1989) Radiotoxicity of 5-[123I]iodo-2'-deoxyuridine in V79 cells: a comparison with 5[125I]iodo-2'-deoxyuridine. Radiat. Res., 118, 532–544. Martin RF and Haseltine WA (1981) Range of radiochemical damage to DNA with decay of iodine-125. Science, 213, 896–898. Netti PA, Hamberg LM, Babich JW, Kierstead D, Graham W, Hunter GJ, Wolf GL, Fischman A, Boucher Y and Jain RK (1999) Enhancement of fluid filtration across tumor vessels: implication for delivery of macromolecules. Proc. Natl. Acad. Sci. USA, 96, 3137–3142. Nikula TK, McDevitt MR, Finn RD, Wu C, Kozak RW, Garmestani K, Brechbiel MW, Curcio MJ, Pippin CG, Tiffany-Jones L, Geerlings MW Sr, Apostolidis C, Molinet R, Geerlings MW Jr, Gansow OA and Scheinberg DA (1999) Alpha-emitting bismuth cyclone xylbenzyl DTP A constructs of recombinant humanized anti-CD33 antibodies: pharmacokinetics, bioactivity, toxicity and chemistry. J. Nucl. Med., 40, 166–176. O'Donoghue JA (1994) The impact of tumor cell proliferation in radioimmunotherapy. Cancer, 73, 974980. O'Donoghue JA (1999) Implications of nonuniform tumor doses for radioimmunotherapy. J. Nucl. Med., 40,1337–1341. O'Donoghue JA and Wheldon TE (1996) Targeted radiotherapy using Auger electron emitters. Phys. Med. Biol, 41, 1973-1992. Otte A, Mueller-Brand J, Delias S, Nitzsche EU, Herrmann R and Maecke HR (1998) Yttrium-90-labelled somatostatin-analogue for cancer treatment. Lancet, 351, 417–418. Palm S, Back T, Claesson I, Delle U, Hultbom R, Jacobsson L, Kopf I and Lindegren S (1998) Effects of the alpha-particle emitter At-211 and low-dose-rate gamma-radiation on the human cell line Colo-205 as studied with a growth assay. Anticancer Res., 18, 1671–1676. Pomplun E, Booz J and Charlton DE (1987) A Monte Carlo simulation of Auger cascades. Radiat. Res., 111, 533–552. Porteous DD (1971) The toxicity of 125IUdR in cultured mouse BP8 tumour cells. Br. J. Cancer, 25, 594597.

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28. RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION DAVID R. VERAA, CARL K. HOHA, ROBERT C. STADALNIK8 AND KENNETH A. KROHNC A

Department of Radiology, University of California, San Diego School of Medicine, 200 West Arbor Drive, San Diego, CA 92103, U.S.A.;BDepartment of Radiology, University of California, Davis School of Medicine, 4860 Y Street, Sacramento, CA 95817, U.S.A.; and c Department of Radiology, University of Washington School of Medicine, Seattle, WA 98195, USA.

INTRODUCTION The purpose of this chapter is to illustrate a concept of functional imaging using the study of liver and renal function as examples. We will not attempt a complete review of liver and renal radiopharmaceuticals nor will we catalog clinical or experimental radiotracers for these organs. The agents and methods are well established and have been extensively reviewed (Krishnamurthy & Krishnamurthy, 2000; Tauxe, 1995; Dubovsky, 1985; Sandier et al., 1996), but the rich history of liver and kidney agents motivates the theme for this chapter. Our goal is to develop a paradigm by which new radiopharmaceuticals for the evaluation of tissue function may be successfully developed. We propose the hypothesis that a successful radiopharmaceutical should measure at least two physiologic or biochemical processes that satisfy two criteria. First, each process must be sensitive to tissue pathology. And second, each process must alter the radiopharmaceutical's uptake kinetics in a unique fashion. Our motivation for this proposal is based on the fact that diagnostic agents and/or imaging modalities exist for most organ systems, especially for the liver and kidney. Consequently, a new imaging agent should provide a significant increment in diagnostic information. This strategy should lead to new radiopharmaceuticals that are responsive to multiple diseases. This is the rationale for our requirement that the radiotracer be sensitive to at least two biochemical processes and that each pathway be uniquely altered by a specific disease state. The key to this proposal is that each disease must alter the tracer's biodistribution in a manner that will identify each disease. Our hypothesis is independent of the identification method. The protocol may be as simple as two blood samples at specific times post-injection, or two images acquired at different times post-injection. A more complex method could require a dynamic scan for 30 minutes followed by a kinetic analysis of the timeactivity data. We will provide examples of liver and kidney protocols that utilize these various schemes. This chapter will be organized conceptually to first illustrate the requirement for unique sensitivity of disease states. Next we will illustrate this concept using the liver and kidney as examples. For each organ we will

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discuss the major physiologic and biochemical pathways that may be altered in disease and men consider relevant functional measurements. CHEMISTRY AN ORTHOGONAL PROCESS The second requirement of our hypothesis is called orthogonality. It is a general concept, which refers to two events that do not influence each other. Orthogonal processes are statistically independent and exhibit zero correlation. A linear regression of two orthogonal variables will result in a correlation coefficient of zero. SOME PHYSIOLOGIC AND BIOCHEMICAL VARIABLES The time course that a tracer follows through some region of tissue reflects numerous physiological and biochemical processes. Table 1 divides these processes into four categories: delivery of the tracer, regional extraction by the target cells, intracellular processing, and return to the circulation and eventual elimination. The radiopharmaceutical chemist has a range of tools available to modify each process. These include how the radiopharmaceutical is constructed and non-radioactive drugs that might be administered as part of the nuclear medicine procedure. For example we can alter the size and charge on a molecule to change protein binding or affinity for a receptor or one can introduce electronic changes to specifically block a step in the metabolism of a radiolabeled drug. The classic example of this is FDG where substitution of an -OH group with the isoelectronic -F group totally inhibits the isomerase enzyme (Wick et al., 1957; Gallagher et al., 1978). Likewise the co-administration of non-radioactive drugs, especially carrier, can change the time course of a radiopharmaceutical. Here the classic example is the growing appreciation that carrier-added receptor studies are often more useful than high specific activity studies. In the latter case the biodistribution kinetics at high specific activity is used to separate flow parameters from regional receptor binding potential but the biodistribution kinetics at low specific activity can be used to separate the forward binding rate constant from the receptor concentration (Vera et al., 1991). Table 1. Physiological and Biochemical Processing of a Radiotracer • Delivery of the tracer - As a single substrate or bound to a macromolecular carrier - True substrate or intravascular metabolite from other site • Regional extraction by target cells - into an interstitial space - binding to the target molecule - endocytosis • Intracellular processing - metabolism ° phosphorylation leading to retention ° conjugation leading to excretion - catabolism leading to excretion • Return to circulation and eventual elimination

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MEASUREMENT OF LIVER FUNCTION HEPATIC FUNCTION A useful general description of the liver is given by Arias et al., who classified its structural organization in terms of "its special function as a guardian interposed between the digestive tract (and spleen) and the rest of the body"(Arias et al., 1994). To accomplish this role the liver exhibits six unique organizational features. First, the hepatocyte, the functional cellular unit which comprises 85% of the cell population, exhibits both exocrine and endocrine functions. Second, in addition to oxygenated blood from an arterial supply, the liver receives blood directly from the digestive tract via a portal vein. Third, to maximize solute exchange the hepatocytes are arranged in a single-width column along the capillary. Fourth, the capillary-hepatocyte interface lacks a basement membrane, and this permits the interaction of macromolecules at the hepatocyte surface without an interstitial space. Access to the hepatocyte surface by solutes and macromolecules is by the Space of Disse, which is behind large gaps between the endothelial cells lining the hepatic sinusoids or capillary channels. Fifth, the reticuloendothelial system (RES) of Kupffer cells, which comprise the remaining 15% of the cell population, are positioned between the hepatocytes and the blood channels. Sixth, one of the exocrine functions, bile formation, employs a separate lumen to transport metabolites into the digestive tract. Liver Plasma Flow In his rather opinionated text on nuclear hepatology Marshall Brucer emphasized two features of the hepatic blood supply that are pertinent to the interpretation of liver scintigraphs (Brucer, 1977). First is the high variability in liver perfusion. Light exercise or a change in the subject's position will produce changes in hepatic blood flow. This is the result of two factors. The portal vein, which supplies approximately 80% of the blood to the liver, is a low pressure system. Additionally, the liver as well as the digestive tract can alter their plasma volumes almost instantaneously; the result is a rapid change in blood flow. The latter, feature is common to other large vascular organs. The liver sinusoids exhibit a periodicity in blood flow and volume. Brucer cited the classic work of Wakim and Mann, who used a transillumination technique to demonstrate that only one quarter of the hepatic sinusoids actively circulate erythrocytes (Walkin & Mann, 1942). This flow heterogeneity occurs within the hepatic lobule, which is the anatomic functional unit of the liver; a sinusoid within a given lobule may be activity flowing blood, while its neighboring sinusoids are inactive. The switch from active to inactive does not occur at regular intervals. The sequence is random with a cycling time on the order of hours. This feature imposes a serious constraint when designing a liver blood flow agent. Uptake of Intestinal Substances and Redistribution One function of the liver is the uptake of endogenous substances from the intestine, such as endotoxins from intestinal flora. The reticuloendothelial system within the liver phagocytoses foreign cells, such as bacteria. Carbohydrate metabolism occurs within the hepatocyte cytoplasm, and serves to store blood glucose in the form of glycogen. Under modulation by humoral regulatory factors, hepatic enzymes release glucose into the blood by the glycogenolysis pathway. Lipid metabolism in the smooth endoplasmic reticulum of the hypatocytes includes the synthesis of lipids associated with proteins or carbohydrates, the transformation of lipids into chylomicrons (lipoproteins), which are a convenient form of storage and transportation, and the

HANDBOOK OF RADIOPHARMACEUTICALS

798

synthesis of cholesterol. Protein metabolism occurs in the hepatocellular granular endoplasmic reticulum and includes the synthesis of plasma proteins, such as albumin, globulins, fibrinogen, and prothrombin, LDL uptake, glycoprotein uptake and transport to the lysosomes for catabolism and conservation of amino acids. Adjacent hepatocytes indent to form bile canaliculi, where the cells excrete salts and biliary pigments, such as bilirubin. Biotransformation ofXenobiotics The hepatocyte employs both oxidative and nonoxidative pathways to detoxify foreign compounds, with eventual elimination via the biliary or the urinary route. The general mechanism is the transformation of lipid-soluble substances to water-soluble conjugates. The most common pathways are glucuronidation and sulfation. Catabolism of lipophilic drugs and steroid hormones occurs in the smooth endoplasmic reticulum. EVALUATION OF LIVER FUNCTION A radiopharmaceutical R* administered into the extra-hepatic plasma volume is delivered into the hepatic plasma by hepatic arterial and portal plasma flow (Fa and Fp, respectively). This scheme is illustrated in Figure 1 using the symbols defined in Table 2. The rate at which the radiopharmaceutical enters the hepatic plasma volume is governed by the magnitude of (Fa + FP)/VP (Krohn et al, 1982). Once in the hepatic plasma, the agent can be extracted by hepatocytes (process kh) or Kupffer's cells (process kk). If the agent is a receptor-binding radiopharmaceutical, the rate of kh or kk will be governed by the concentration of a specific receptor [R]o and the associated forward binding rate constant k. If the hepatic extraction is low, some of the agent can exit the liver and return to the extra-hepatic blood. The rate at which the radiopharmaceutical exits the hepatic plasma volume is governed by the magnitude of (Fa + Fp)/Vhp. The hepatocyte can metabolize the agent and excrete the labeled metabolite into the bile (kmb) or blood, where it will accumulate (process kmu) in the urinary system. Hepatic radiotracers and the processes that they trace are listed in Table 3.

ExtraHepatic Plasma

Hepatic Plasma

W

"P

Hepatocyte F1h

[Rip

W

^

4

(F.+FJ/VL

[R1hp "hn

Biliary System

k

mb

^

F1b

h

Flu

^ fc

k k

PI* 1

Kupffer Cell

mu

Urinary System

Figure 1. Schematic diagram illustrating the physiologic and biochemical pathways pertinent to hepatic radiotracers. See Table 2 for symbol descriptions.

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION

799

Table 2. Symbol Table for Figure 1 Symbol Fa Fp Vp Vhp kh kk kmb kmi,

Process Hepatic Arterial Plasma Row Hepatic Portal Plasma Flow Extra-Hepatic Plasma Volume Hepatic Plasma Volume Hepatocyte Extraction Kupffer's Cell Extraction Metabolism and Biliary Metabolism and Excretion

Hepatic Perfusion Tc-99m sulfur colloid ("Tc-SC) was developed at Brookhaven National Laboratory by Richards and coworkers (Harper et al,, 1964). The original method, which produced sulfur particles by bubbling hydrogen sulfide gas through an acidified solution of sodium pertechnetate and gelatin, yielded particles of 0.08 to 0.10 microns in size.

The thiosulfate-based kit, simultaneously introduced in 1966 by Stern, McAfee and

Subramanian, Larson and Nelp, Atkins et al., and Patton et al., remains the most convenient synthesis and the basis for the commercial kits available today(Stern et al., 1966; Larson & Nelp, 1966; Atkins et al,, Patton et al., 1966). This method produces particles with a size distribution from 0.1 to 1.0 microns (Davis et al., 1974). Technetium~99m phytate (Subramanian, et al, 1973) is injected as a solution of technetium-99m stannous inositol hexaphosphate and combines with plasma and tissue calcium to form a true colloid with particles less than 0.01 microns. The larger particles exhibited faster blood clearance half-times; thiosulfatebased 99mTc-SC typically clears the blood with a half-time of 4 minutes in a healthy subject and smaller (< 0.1 micron) particles typically clear with a 6-minute half-time (Brucer, 1977). The exception to this rule is Tc-99m phytate, which has a larger initial distribution volume and therefore exhibits clearance times that are typically three-fold longer. Measurement of hepatic blood flow preceded the development of liver imaging. Phosphorus-32 chromate phosphate colloid was introduced for the measurement of hepatic blood flow by Dobson and Jones(Dobson & Jones, 1949). As an alternative, Vetter et al. introduced radioactive gold colloid and reported a 0.8 extraction coefficient in healthy human livers (Vetter et al., 1954). They concluded that radio-gold colloid underestimated hepatic blood flow by 20%. Later that same year, Cassen and co-workers (Stirrett et al., 1954) published the first liver images using l98Au-colloid. During these early years investigators considered radioactive colloids as hepatic clearance agents and developed a set of criteria for their design (Dobson et al., 1966). With the goal of measuring hepatic blood flow, the chief design parameter was the size distribution of the colloidal suspension.

800

HANDBOOK OF RADIOPHARMACEUTICALS Table 3. Liver Radiopharmaceuticals Radiopharmaceutical iy8 Au-Colloid "•"Tc-SulfurCoUoid 131 I-Rose Bengal "Tc-IDA "Tc-NGA

Process F F

Although, phosphorus-32 and gold-198 are no longer used as clinical or investigational liver function agents, their legacy still pervades the terminology and conceptual basis for our current liver imaging agent, "TcSulfur Colloid. First, unlike the radio-phosphorus and radio-gold agents, "Tc-SC is not a true colloid; the particles are outside the size range required of a colloidal suspension. As a result, the particles settle upon standing; a true colloidal suspension will maintain a homogenous solution. Second, mediation of serum opsonins (Saba, 1982) has been demonstrated for l98Au-colloids (Wagner & lio, 1964), but not for large particles, such as "Tc-SC. Third, the technetium-99m colloids have inherited the explanation for hepatic uptake from gold-colloid. Nuclear Medicine textbooks glibly attribute hepatic localization of 99mTc-Sulfur Colloid to Kupffer cell uptake. For example, Oppenheim refers to phagocytosis by the RES (Oppenheim, 1996) and Pinsky and Johnson refer to opsonin-mediated uptake by Kupffer cells (Pinsky & Johnson 1984). In actuality, the mechanism of hepatic99mTc-SCuptake is unknown. Most of the evidence for RES uptake is indirect, such as the correlation of organ percent-of-injected dose with percent of cardiac output (Help, 1975). One of the few direct investigations (Chaudhuri et al., 1973) of99mTc-SClocalization was carried out by autoradiography of liver tissue samples. Localization of the technetium-99m-labeled particles was not confined to Kupffer cells; this was in contrast to the gold-198 colloid tissue samples where the activity was confined to the Kupffer cells. The most likely uptake mechanism, proposed by Brucer, is entrapment within the Space of Disse (Brucer, 1977). The last inheritance from gold colloid pertains to the physiologic interpretation of 99mTc-SC images. Textbook discussions of "Tc-SC provide instances where liver image interpretation is based on RES function. Examples are end-stage cirrhosis and fulminate hepatitis, where hepatic function is impaired and 99m Tc-SC uptake by the spleen and bone marrow is increased. The proposition that99mTc-SCuptake can be limited by the number of RE cells is not based on direct evidence. The only evidence is based on gold colloid and albumin microaggregates. Using l98Au-colloid, Cohen et al. demonstrated in rats that increasing the number of particles per body weight decreased the rate of clearance from the blood (Cohen, et al., 1968). Atkins and coworkers observed changes in the liver, spleen, and marrow distribution with increasing doses of H2S- and thiosulfate-generated99mTc-Sulfur Colloid; they did not, however monitor blood disappearance or organ uptake (Atkins et al., 1970). lio et al demonstrated uptake sensitivity of "RES functional capacity" in

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION dogs and humans using sensitivity of

801

131

I-microaggregates of albumin (Iio et al., 1964). Although unsubstantiated, kinetic

99m

Tc-SC hepatic uptake to tissue mass or function may be real. During end-stage liver disease,

which is when this phenomenon is observed, the number of Kupffer cells or the volume within the Space of Disse may limit the number of 99mTc-SC particles extracted by the liver. The result would be more effective competition by the spleen and bone marrow for 99mTc-SC uptake. During the past decade computed tomography (CT) has replaced

99m

Tc-Sulfur Colloid scanning for liver

tumors (Paley & Ros, 1998). Helical CT can provide volumetric scans of the entire liver during a single breath-hold. The result is a dramatic improvement in spatial resolution by holding the lesions motionless during image acquisition. Respiration during a 10-minute SPECT study produces up to 2 cm excursions in liver motion. Perhaps the most enduring legacy of 99mTc-Sulfur Colloid is the hepatic angiogram (Stadalnik et al., 1975), which forms the underlying concept for biphasic contrast-enhanced CT (Oliver & Baron, 1996). Both techniques use the fact that most malignant neoplasms require more oxygenated blood, and therefore, receive a high proportion of their blood from the hepatic artery rather than the portal vein. Consequently, hepatic metastases and hepatomas accumulate radioactivity or contrast agent earlier than the surrounding tissue. This occurs because the activity within the portal blood must travel through the gastrointestinal tract before arriving at the liver. Thus, arterial and portal phase images are acquired and compared. Each phase (or image) represents a different cardiovascular process, and these are independently controlled by the pathophysiology of the tumor. These two processes are depicted in Figure 1 as Fa /Vp and Fp/Vp. This, our first example of an imaging protocol based on orthogonal measurements, has served as a valuable diagnostic concept for over 25 years. Biliary Excretion Excretion of a radiotracer into the hepatic biliary system provides an opportunity to measure two processes, the excretion rate and the rate at which the liver extracts the agent from the blood. Figure 1 depicts these processes as kh and kmb, respectively. The simplest means to document each process would be an early image of the liver during the extraction phase and a late image during the excretion phase. If extraction of the agent is high, for example greater than 90% during a single pass through the liver, then the extraction phase will be a measure of hepatic blood flow. However, if the extraction is low, the uptake phase would provide a measure of the biochemical process (kh) by which the hepatocytes recognize and bind the radiotracer. Because the hepatobiliary agents presented in this section exhibit a low hepatocyte extraction fraction, the agent is sensitive to the hepatocellular uptake mechanism. In general, because both processes are operating in opposite directions, extraction and excretion phases can be considered orthogonal. Consequently, two or perhaps four properly timed images or a dynamic imaging study can independently measure each process. The first hepatobiliary agent,

131

I-Rose Bengal, was developed by Taplin and coworkers in 1955 for the

measurement of hepatocellular function (Taplin et al., 1955). In an effort to increase the sensitivity oi blood clearance measurements, Taplin introduced the concept of a "stress" test. Knowing that another dye, bromosulfophthalein (BSP), competed with rose bengal for hepatic uptake, Taplin coinjected non-radioactive

802

HANDBOOK OF RADIOPHARMACEUTICALS

BSP with the radiopharmaceutical. This slowed the clearance from the blood by healthy livers, but significantly prolonged the clearance in patients with cirrhosis. Without the BSP competition, the blood halftime for healthy patients and cirrhosis would be similar. Two serious deficiencies prevented the wide-spread use of 131I-Rose Bengal. First, was the quality and quantity of the photon emission provided by 131I, where the high gamma energy emission was not compatible with the low energy requirements of the new gamma camera developed by Anger (Anger, 1958; Anger, 1964). The second limitation was the inhibition of hepatic uptake by serum bilirubin, which restricted its value in jaundiced patients. Hepatic uptake of rose bengal is mediated by the same an ionic clearance mechanism as bilirubin and BSP. This is a high capacity transport system at the hepatocyte plasma membrane (Fortner, 1977). Anionic dyes, such as rose bengal, BSP, and bilirubin are stored in the cytosol by the y and Z proteins. This also is a high capacity system. After conjugation, bilirubin and the unconjugated dyes are transported across the hepatocyte-bile canalicular interface. This is a low capacity process that requires active transport across a high concentration gradient. As a result of this down-stream competition, the excretion phase is more sensitive to bilirubin. Blood clearance and liver accumulation will be unaltered, but the rate of elimination from the hepatocytes and accumulation in the biliary tree is significantly depressed. At higher serum bilirubin levels the extraction phase is affected, which prolongs blood clearance and hepatocellular accumulation. Advances were made in hepatobilary agents using the dosimetric and imaging advantages of Tc-99m. In 1975, Baker and co-workers developed "Tc-pyridoxylidene glutamate (99mTc-PyG) by autoclaving monosodium glutamate, pyridoxal hydrochloride, and sodium 99mTc-pertechnectate (Baker et al., 1975). The resulting Schiff base complexed 99mTc via the pyridoxal and glutamic acid. Jansholt and co-workers (Jansholt et al., 1978) used HPLC to demonstrate that the autoclave method produced multiple radiochemical products. A method for the technetium labeling of pyridoxylidene amino acids was developed by Kato and Hazue using tin reduction at room temperature (Kato & Hazue, 1978). This method produced a single radiochemical, with a single peak on HPLC. In 1982 Kato-Azuma reported the development of a superior hepatobiliary radiopharmaceutical, 99mTc-N-pyridoxyl-5-methyltryptophan (99mTc-PHMT), which produced a stable amine linkage from condensation of the pyridoxyl aldehyde group and the tryptophan amino group (Kato-Azuma, 1982). The product, which demonstrated rapid hepatobiliary kinetics and low inhibition by serum bilirubin, is the current hepatobiliary agent available in Japan. Initial patient studies using "Tc-PyG were reported by Ronai and co-workers in 1975 (Ronai et al., 1975). In the United States initial patient studies were conducted by Stadalnik and colleagues in 1976 (Stadalniket al., 1976). The class of hepatobiliary agents, technetium-99m-labeled iminodiacetic acids ("Tc-IDA), used today in the U.S. and Europe was serendipitously discovered by Loberg and co-workers in 1976 during a search for a technetium-99m-based myocardial agent (Loberg et al., 1976). The agent was 99mTc-N (2,6dimemylphenylcait»amoylmethyl)-iminodiacetic acid (99mTc-HIDA) and was initially intended as a labeled lidocaine analog. In vivo competitive clearance studies in dogs with co-injected BSP confirmed (Harvey et al., 1979) that 99mTcHIDA was transported through hepatocytes by the organic-anion pathway. Structural studies (Loberg & Fields, 1979) demonstrated that "Tc-HIDA existed in solution as a bis structure with a net charge of -1 and with

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION

803

the technetium in the +3 oxidation state. Neither stannous nor stannic ion was present in the final radiotracer complex. The dimeric product, which was unexpected, was consistent with the molecular weight required for hepatic clearance (Firnau, 1976); the molecular weight of monomeric N (2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid is below the required range. The technetium-99m-labeled IDA agents offered two advantages over radiolabeled dyes. First, they were technetium-99m-based radiopharmaceuticals, and therefore, optimized for gamma scintillation detectors. And second, chemical modification to the aromatic ring of the HIDA permitted optimization of clinical performance. By 1979 the consensus was that the optimum use of 99mTc~IDA agents would be the assessment of cystic duct patency for the diagnosis of acute cholecystitis (Loberg et al., 1979). This goal defined the ideal properties to be 1) rapid extraction from the plasma, 2) effective competition with bilirubin, 3) rapid transit through the hepatocyte, 4) high biliary concentration, and 5) low renal excretion. In addition to HIDA with its ortho-dimethyl substitutions, four other IDA derivatives were tested (Wistow et al., 1978): 2,6-diethylacelanilido-IDA, 4-isopropylacetanilido-IDA, 4-butylacetanilido-IDA, and 2,6-diisopropyl acetanilido-IDA. The parabutyl substitution exhibited the highest plasma protein binding and the lowest renal excretion. It also demonstrated in animals (Jansolt et al., 1980) and humans (Fonseca et al., 1979) the most effective competition with bilirubin, but, unfortunately, the slowest hepatocyte transient time. The 2,6-diisopropyl derivative, 99mTc-disofenin (Hepatolite) exhibited the best combination of rapid hepatocyte transit time and low bilirubin inhibition (Stadalnik et al., 1981). Using structure-biodistribution analysis based on the correlation of renal elimination and reverse-phase HPLC, Nunn proposed 3-bromo-2,4,6-trimethyl-IDA (99mTc-mebrofenin, Choletec)(Nunn, 1983). This agent also exhibited rapid hepatocyte transit time and low bilirubin inhibition (Nunn et al., 1983). Hepatocellular Functional Mass Imaging of hepatocellular function employs a new class of radiotracers called receptor-binding radiopharmaceuticals (Eckelman et al., 1979). These agents, by virtue of their chemical structure, are recognized and bound by a specific target molecule called a receptor. The general strategy is to select receptors that are specific to a target tissue or tumor type, and then develop a labeled analog to the native receptor ligand. The target receptor used for hepatic imaging was discovered in the late 1960s when Ashwell, Morell, and coworkers (Morell et al., 1968) investigated the biodistribution of ceruloplasmin by radiolabeling its carbohydrate side-chain. The receptor was initially called hepatic binding protein (HBP) (Stockert & Morell, 1983) and later the asialoglycoprotein (ASGP) receptor (Steer, 1996). The receptor recognizes and binds glycoproteins with carbohydrate side-chains that terminate in galactose.

The physiologic role of this

receptor, which is exclusive to hepatocytes, is the homeostasis of plasma glycoproteins (Ashwell & Steer, 1981). This is based of the fact that all plasma proteins, with the exception of albumin, are initially synthesized by the liver and excreted into the blood with carbohydrate side-chains that terminate in sialic acid. Because sialic acid forms weak linkages to other carbohydrates, it is not stable within the plasma. Depending on the glycoprotein and time within the plasma, there is a probability that the sialic acid moiety will be cleaved. This exposes the penultimate sugar in the chain, which is galactose. When this occurs the

804

HANDBOOK OF RADIOPHARMACEUTICALS

receptor extracts the protein from the blood and transports it to the hepatocellular lysosomes, where it is catabolized into amino acids. Consequently, imaging the liver via the ASGP receptor traces the cellular biology of plasma protein homeostasis and hepatic amino acid turnover. Introduction of the first labeled ASGP receptor analog was in 1979. After imaging with 123 I-asialoceruloplasim, a native ligand, Vera, Krohn and Stadalnik synthesized an analog ligand by covalent attachment of galactose to human serum albumin (HSA)(Ver. et al., 1979). The resulting neoglycoconjugate was called galactosyl-neoglycoalbumin (NGA). In vivo biodistribution studies of "Tc-NGA in rabbits demonstrated dose dependent uptake (Vera et al., 1984a). Additionally, the rate of hepatic uptake could be controlled by the galactose density, the number of galactose units per HSA, of the NGA. This dependency led to the modulation of the ligand-receptor affinity by the galactose density (Vera et al., 1984b), which could be chemically controlled during the coupling of the galactose ligands to the albumin backbone (Vera et al., 1985a). Dynamic imaging studies in pigs later demonstrated kinetic sensitivity to hepatic plasma flow, as well as NGA molar dose and receptor-ligand affinity (Vera et al., 1989). The ability of the molar dose to control the shape of the time-activity data, implied that the amount of receptor within the target tissue could also control the rate of "Tc-NGA accumulation. This was later confirmed based on a significant correlation of receptor density with an index of "Tc-NGA-hepatic uptake (Kudo et al., 1991 a). The receptor density was measured by in vitro binding assay of hepatic biopsy samples obtained after the 99m Tc-NGA studies. The first clinical trial with "Tc-NGA (Stadalnik et al., 1985) confirmed two important features of ASGP receptor-based imaging (Stadalnik et al., 1993). The first feature, which was predicted by the known molecular biology of the ASGP receptor, was the lack of inhibition by elevated levels of serum bilirubin. The second feature was the absence of accumulation by hepatocellular carcinomas (HHC) and metastases. This uptake pattern was later confirmed by Virgolini and coworkers (Virgolini et al., 1990). Differentiation of HHCs from metastases was achieved by an injection of l23I-Tyr-insulin (Kurtaran et al., 1995). Having a 1000-fold concentration insulin receptor over normal tissue, HCC accumulated the labeled insulin. Metastases, which do not express the insulin receptor, remained devoid of radioactivity. Later studies at the University of Vienna demonstrated a normal or even increased uptake by focal nodular hyperplasia (Kurtaran et al., 1997), a finding which was also consistent with the known cellular biology of the ASGP receptor. The "Tc-NGA protocols commonly use a dose that is scaled to the patient's body weight, 1.8 nanomole per kilogram. In 1992 a DTPA-conjugate of NGA was introduced in Japan as a commercial product (Torizuka, et al. 1992). The attachment of a chelator, typically 4 per HSA, permitted the formulation of a tin-based freeze-dried kit, which could be labeled within 30 minutes by the simple addition of sodium pertechnetate. Named Asialoscinti, or99mTc-GSA,it was the first commercially-available receptor-binding radiopharmaceutical. To provide a quantitative index of hepatic function Kudo and co-workers proposed the LHL15 parameter, which was the ratio of the counts within the liver region-of-interest (ROI) over the sum of the counts within the liver and heart ROIs (Kudo et al., 1991c). This parameter provided significant correlations with serum albumin and bilirubin concentrations, prothrombin times, indocyanine green retention at 15 minutes (ICG

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION

805

R15), Child-Turcotte criteria (CTC) scores. The 99mTc-GSA protocol uses a 41-nanomole dose, independent of the patient's body weight. The initial intention was to simultaneously measure hepatic plasma flow F, ligand-receptor affinity kb, and receptor concentration [R]0 (Krohn et al., 1982).

The goal was to employ the receptor concentration

measurements as an index of hepatic functional reserve and to classify disease based on the magnitude of each parameter (Stadalnik et al., 1986). An engineering technique, local parameter identifiability analysis, was employed to optimize the

99m

Tc-GSA imaging protocol. Using computer simulations of the 99mTc-NGA

radiopharmacokinetic model (Vera et al., 1985b), it was determined that the three parameters F, kb, and [R]0 could be independently measured if the imaging protocol employed a 99mTc-NGA of moderate affinity (30 galactose per HSA), a single blood sample, both the liver and heart time-activity data, as well as administration of a molar dose capable of occupying at least 50% of the receptor. Computer programs for measurement of parameters F, kb, and [R]0 have been implemented for the 99mTc-NGA imaging protocol (Vera et al., 1991) and the99mTc-GSA imaging protocol (Kudo et al, 1990; Ha-Kawa & Tanaka, 1991; Miki et al., 1997). The Ha-Kawa and Miki programs do not require a blood sample. Using 99mTc-GSA, Kudo and co-workers demonstrated a significant correlation of [R]0 with conventional liver function tests (Kudo et al., 1993). Pimstone and co-workers at U.C. Davis (Pimstone et al., 1994) reported a high correlation of [R]0 with the aminopyrine breath test (ABT) (Galizzi et al., 1978), a measure of hepatic microsomal function, and the Pugh-modified CTC score (Pugh et al., 1973). Using the U.C. Davis (Vera et al., 1991) computer program and 99mTc-NGA imaging protocol, Virgolini and co-workers at the University of Vienna demonstrated reduced [R]0 measurements in cancer-bearing livers (Virgolini et al., 1990) and hepatitis (Virgolini et al., 1992). As a final step in the confirmation of 99mTc-NGA functional imaging, Kudo and coworkers (Kudo et al., 1991b) demonstrated that the computer modeling measurements of [R]0 were plausible; [R]0 values showed good correlation with measurements by in vitro assay of liver biopsy samples obtained immediately after 99mTc-NGA functional imaging. Receptor-targeted imaging via 99mTc-NGA and 99mTc-GSA has been applied to liver transplantation. Woodle and co-workers (Woodle et al., 1989) observed a good correlation of hepatic allograft function with 99mTcNGA images and kinetics.

Sakahara and co-workers at Kyoto University employed a Patlak graphical

analysis to measure graft function after auxiliary partial orthotopic transplantation (Sakahara et al., 1999). They concluded that ASGP receptor scintigraphy was useful for distinguishing and monitoring graft and native liver functions. Using the Miki-99mTc-GSA model, investigators at the University of Tokyo (Kita, et al., 1998) monitored allograft functional reserve by [R]0. Recent studies have demonstrated an ability of 99mTc-GSA imaging to reflect changes in hepatic function after placement of a transjugular intrahepatic portosystemic shunt (TIPS), which produces a decrease in the portal blood supply to the liver. When patients were imaged before and after TIPS, the LHL15 values decreased (Kira, et al., 1997); the greatest decrease in 99mTc-GSA uptake occurred in subjects with a poor post-TIPS prognosis. Stein and co-workers demonstrated a diminished [R]0 after TIPS in pigs (Stein et al., 2000). The receptor concentration did not return to the pre-TIPS values after occlusion of the stent. This confirmed that the diminished uptake was not due to hepatic shunting, but was the result of diminished

806

HANDBOOK OF RADIOPHARMACEUTICALS

functional reserve, presumably due to a loss of portal blood and the nutrients that it delivers to the liver. A similar loss of receptor function was measured in the rats with a chronic portosystemic shunt (Colquhoun et al., 2001). ASGP receptor density of the shunted rats was significantly lower than rats with a sham operation. Liver histopathology of the shunted group remained normal and conventional liver function tests, with the exception of serum ammonia levels, also remained unchanged between the shunted and sham groups. This result suggested that the in vivo receptor measurement was more sensitive than histology or standard liver function tests. In Japan the most common indication for 99mTc-GSA imaging is the measurement of hepatic functional reserve prior to hepatectomy (Kokudo et al., 1999). This is based on the assumption that receptor concentration measurements and static liver images are an index of liver functional mass. This was demonstrated for receptor measurements (Miki et al., 2001) using kinetic modeling of "Tc-GSA. The ASGP-receptor amount, as calculated by [R]0Vhp, exhibited a significant correlation with total hepatocyte number, which was measured by histomorphometry. Based on radiopharmacokinetic analysis of 99mTc-GSA functional imaging studies in 90 patients, Kwon and co-workers concluded that measurement of a subject's maximal 99mTc-GSA removal rate Rmax was useful for selecting candidates for hepatectomy and that di- and tri-segmentectomies were high-risk procedures when Rmax was below 0.35(Kwon et al., 1997). Technetium99m-GSA is also used to assess patients for hepatic transplantation by establishing a prognosis with the native liver. LHL15 values accurately predicted 1-year survival in patients with inoperable HCC and cirrhosis (Takeuchi et al., 1999). Based on a regression analysis with Cox's proportional hazards modeling Saski and co-workers demonstrated a significant correlation with survival rate (Saski et al., 1999). Single photon emission computed tomography (SPECT) can be employed with 99mTc-GSA (Ichihara et al., 1997). Dynamic SPECT has been used to construct Patlak plots from regional time-activity data (Hwang et al., 1999). Matsuzaki and co-worker performed SPECT 99mTc-GSA studies of patients with chronic liver disease and healthy controls(Matsuzaki et al., 1997). Base on the left-to-right lobe ratio of the imaging and intact hepatocyte theory, they concluded that, during the onset of cirrhosis, the reduction of hepatic functional reserve per unit hepatic volume, is slower in the left lobe. To this point we have presented the use of a single functional measurement, the receptor concentration [R]0. Although effective as a measure of hepatic functional reserve, it alone cannot classify the disease state of the liver. A second parameter would be required to uniquely classify the functional state of the tissue. For example (Vera et al., 1979; Stadalnik et al., 1986) a liver with a decreased hepatic blood flow and a decreased receptor concentration would be classified as cirrhotic, or an increased hepatic blood flow and a decreased receptor concentration would be classified as hepatitis. Based on identifiability analysis of the 99m Tc-NGA radiopharmacokinetic system (Vera et al., 1985), scaled hepatic blood flow F/Ve and receptor concentration [R]0 can be simultaneously estimated with good precision and can, therefore, be considered orthogonal. The last criteria for a successful classification scheme is that each physiologic or biochemical parameter must be diagnostic. Figure 2 is a receiver operating characteristic (ROC) analysis of TcNGA kinetic modeling parameters. It can be used to determine if the parameters are diagnostic. Unfortunately the ROC curves demonstrate the inability of the forward binding rate constant kb or hepatic perfusion F/Ve to

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION discriminate healthy subjects from patients with liver disease.

807

A diagonal ROC curve indicates a

discrimination probability equal to a coin toss. Note that the receptor concentration [R]0 exhibited a very high diagnostic accuracy (Vera et al., 1996), which is indicated by a large area under the ROC curve.

1 0 ROC Analysis o/ / X 2/ X X X -

0.8 t / /

'5 .*— ( 0.6

•:

1 0.4 CO

3

6

/

/ PA/

A^ X

/X

/,/

— a — [R]0 • —o— ^ 0.2 A 7 C/. "Ct\ T • / ~ A r/V // ^" 00 « j , , , i . . i i , . . i , , , i , , , ;

0 0 0.2 0.4 0.6 0.8 1 0

1 - Specificity Figure 2. A receiver operating characteristic (ROC) analysis of TcNGA kinetic modeling parameters.

MEASUREMENT OF RENAL FUNCTION RENAL FUNCTION The kidney selectively removes toxins from the body while at the same time retaining body water and desirable solutes.

The kidney also serves as an endocrine organ by secreting hormones into the blood for

stimlulating the bone marrow to manufacture red blood cells (erythropoetin) and for regulating the systemic blood pressure (renin). Renal Plasma Flow Basic renal physiology can be modeled with concepts commonly used in chemical engineering. The model of the nephron, the basic physiologic unit of the kidney, is shown in Figure 3. The first component of the nephron is the glomerulus, which is a unidirectional filter. The glomerular capillary retains solutes with large molecular size (>44 A) while allowing small solutes and plasma to pass through into Bowman's capsule. However, other solute characteristics are also important in the ability to pass into Bowman's capsule, such as molecular configuration, and electronic charge. The fluid in Bowman's capsule will eventually form urine and be eliminated from the body. In a normal nephron, about 20% of the total plasma entering the kidney is filtered (filtration fraction = 0.2) and appears in Bowman's capsule. Although the mechanical sieve is an

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HANDBOOK OF RADIOPHARMACEUTICALS

oversimplification of the glomerular capillary, it provides a useful conceptual model for understanding the current radiopharmaceuticals. Glomerular Filtration The rate of filtration of solute and fluid through this filter is dependent on the difference in total pressure across the glomerular membrane, the capillary permeability, and the size of the capillary bed. The total pressure of either side of the membrane includes the hydrostatic pressure and the oncotic pressure. The hydrostatic pressure can be controlled by microvascular vasoconstriction or relaxation of the afferent arteriol (before the glomerular capillary) or efferent arteriol (after the glomerular capillary).

For example,

glomerular filtration rate can be increased by simply increasing the vasoconstriction of the efferent arteriol. On the other hand, disease and physiologic conditions which may decrease the filtration rate include dehydration, systemic hypotension, high non-filtered solutes (high oncotic pressure), and intrinsic renal diseases, which may decrease the membrane/filtration surface area (glomerular nephritis, glomerular sclerosis). Small radiopharmaceuticals, such as 99mTc-diethyenetriaminepentaacetic acid (99mTc-DTPA) can serve as tracers of glomerular filtration. Because the kidneys have collateral circulation, which supplies nutrients to connective tissues and other kidney tissues, not all renal blood flow leads to a nephron. Therefore even an ideal tracer for blood flow (high extraction fraction, low metabolism, and reversible plasma protein binding) will underestimate the true renal blood flow. Tubular Secretion and Tubular Reabsorption The next components after the glomerulus are the proximal convoluted tubule and the post-glomerular capillary. The proximal convoluted tubule contains the filtered fluid, with both undesirable and desirable solutes.

The post glomerular or peritubular capillary is a second capillary system in series after the

glomerular capillary and contains blood with the unfiltered solutes. There is a complex exchange of solutes between the blood in the peritubular capillary system and fluid in the proximal convoluted tubules, that is designed to conserve useful body constituents. Most of the tubular water (75%) is reabsorbed back into the blood. Ions such as Cl- and HCO3- are almost completely absorbed along the length of the proximal tubules. In normal renal physiology, the proximal tubule also recovers important body solutes found in the filtrate (amino acids, ascorbic acid, ketone bodies, uric acid, electrolytes [Na+, K+, PO 4 , SO4] and glucose. Interestingly, the commonly used PET radiopharmaceutical, l8F-fluorodeoxyglucose (FDG), remains in the tubular fluid and appears in the urine; it is not transported back into the blood due to its different electronic structure.

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION

Afferent Arteriol

Glomerular Capillary

809 To Renal Vein

Efferent Arteriol

*• ERPR

Bowman'sx. 1 ^/ Capsule y

^ Peritubular Capillary i ubuiar secretion

V

Renal Cortex

Distal Convoluted Tubule

Proximal Convoluted Tubule

Renal Medulla Loop of Henle Vasa Recta

>

-

•••—

VV.J

7 V

^

Increasi ng

•^_— -s/Concent rati on

i

To Collecting System

Figure 3. Schematic diagram showing the basic components of a nephron. Radiopharmaceutials that are filtered enter Bowman's Capsule before becoming concentrated in the proximal tubule. Radiopharmaceuticals that are secreted enter the proximal tubule from the peritubular capillaries. Note that for any tracer to "washout" of the renal cortex, a sufficient glomerular filtration rate (GFR) is needed to "push" the column of tubular fluid through to the collecting system.

Other radiopharamceuticals may get transported in the other direction. For example, iodinated hippuran and Tc~mercaptoacetyltriglycine (99mTc-MAG3) are actively transported (secreted) from the plasma in the post-glomerular capillary into the fluid of the proximal convoluted tubule. Due to the high rate of water reabsorption in the proximal convoluted tubule, any tracer or radiopharmaceutical in the proximal convoluted tubule becomes concentrated. 99m

The next conceptual unit of the nephron is the loop of Henle. Here the tubule containing the filtered solute takes a deep course into the medullary center of the kidney and then returns back to the cortex (Figure 3). There is also a capillary system, the vasa recta, in parallel with the loop of Henle. The purpose of this

810

HANDBOOK. OF RADIOPHARMACEUTICALS

macroanatomical arrangement is because the medullary portion of the kidney has a very high tissue solute concentration. This portion of the nephron utilizes the engineering principle of a countercurrent exchange to efficiently recover even more water.

EVALUATION OF RENAL FUNCTION Figure 4 is a schematic diagram that summarizes the physiologic and biochemical pathways pertinent to a renal radiotracer. The radiopharmaceutical R* is administered into the extra-renal plasma volume where it can flow into the glomerulus. In the glomerulus, a fraction f of the agent is filtered into the proximal tubule. The remainder of the agent ( 1 – f ) will flow into the peritubular capillaries, where the agent can be secreted (process ks,) into the tubules. If the renal extraction is low the agent can exit the kidney and return via the renal vein to the extra-renal blood. Once in the tubule the radiopharmaceutical flows through the distal convoluted tubule and is accumulated (process kpw) in the urinary bladder.

Kidney radiotracers and the

processes that they trace are listed in Table 5.

Renal Cortex Renal Plasma

Glomerulus

Tubule

F

R*

[Rip

f 1

~'l

Bladder V

[R*]t

^

f

F k

s

Peritubular Capillaries Figure 4. Schematic diagram illustrating the physiologic and biochemical pathways pertinent to kidney radiopharmaceuticals. See Table 4 for symbol descriptions.

Table 4. Symbol Table for Figure 4 Symbol F / ks kpw

Process Renal Plasma Flow Glomerular Filtration fraction Tubular Secretion Parenchymal clearance

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION

811

Perfusion An accurate measure of renal blood flow by a relatively non-invasive method provides important clinical information. The assessment of organ perfusion can be approached from two tracer methods. The first method requires that the ideal flow tracer have a high extraction fraction (on a first pass basis through the renal vasculature) and relatively permanent retention in the tissue parenchyma. With this type of tracer, the accumulation (or "integration") of tracer activity early in the study (2-3 minutes) would reflect the blood flow to the kidneys following the Pick principle. Fick's principle states that the plasma flow through an organ of a suitable tracer is directly proportional to the extraction (uptake) by the organ and inversely proportional to the arteriovenous difference of the concentration of the tracer in question across the organ (Welch et al., 1972; Fick, 1870). The important assumption for this type of tracer is that no activity has been lost outside of the kidneys (into urine in the renal pelvis or bladder) since the measurement and calculations are performed early in the study. Also this ideal tracer should not be metabolized by the kidney and needs to rapidly equilibrate between any physiologic compartments within the blood. For example, there needs to be a rapid exchange between plasma and the inside of a red blood cell if there is tracer entry into the red blood cell and rapid equilibrium with plasma proteins if there is any plasma protein binding. Intra-arterial injection of radioactive microspheres is an example of an ideal tracer with high extraction fraction (1.0), no metabolism, and no redistribution. The second type of tracer able to assess organ perfusion is one that has a very rapid diffusion. Radioactive water, 15O-H2O, is an example of a tracer which is not metabolized and has a high diffusion (Inaba et al,. 1989; Juillard et al., 2000). With this type of tracer, a dynamic image acquisition is needed since both the uptake and washout of the tracer in the organ parenchyma is needed to calculate the organ perfusion. Since there is no "integrated" uptake of tracer activity (like in the first method described above) a numerical analysis of organ time-activity curves is needed. Mathematically, this can be done with either deconvolution of the time-activity curve of the tissue or by parameter estimation in a multi-compartmental model. A second PET tracer, 13N-ammonia has also been used for imaging and quantifying renal cortical blood flow (Chen et al., 1992; Nitzche et al., 1993; Killion et al., 1993). The l3N-ammonia has a longer tissue retention time and offers better count statistics than 15O-water, however it has not been validated. Parametric images of renal cortical blood flow have been generated with both 13N-ammonia (Nitzsche et al., 1993) and with 15O-water (Lodge et al., 2000). Effective Renal Plasma Flow As discussed earlier, because a fraction of renal blood flow is detoured around functioning nephron units, even an ideal flow tracer will underestimate the true renal blood flow. Therefore, the term effective renal plasma flow (ERPF) has been used to define the plasma flow which actually comes in contact with the functioning nephrons. Several radiopharamceuticals are available for measuring the ERPF: 131Iorthoiodohippuran (131I-OIH),123 I-orthoiodohippuran, and 99mTc-mercaptoacetyltriglycine (99mTc-MAG3). A new tracer is 99mTc-ethylene dicysteine (99mTc-EC), which is a metabolite of ethylene cysteine dimer (ECD), a brain imaging agent (Kabasakai et al., 2000). While 99mTc-MAG3 has a clearance rate of only 50% of OIH, EC is reported to be 70% of OIH.

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HANDBOOK OF RADIOPHARMACEUTICALS

A variety of quantitative techniques have been reported in the literature for calculating ERPF.

These

methods include deconvolution analysis (Sutton & Kemp, 1993; Gonzales et al., 1994a; Chiwatanarat et al., 1994; Bajen et al., 1997; Flemming & Kemp, 1998), Patlak graphical analysis (Flemming & Kemp, 1998, Shirakawa 1999), discriminate analysis (Gonzales et al., 1994b), and compartmental modeling (Dagli, 1997; Oriuchi et al., 1998). The first compartmental model was executed on an IBM mainframe in 1969 (Jackson & Scheibe, 1969) and was later available clinically (DeGrazia et al., 1974) through AD AC Laboratories.

Table 5. Kidney Radiopharmaceuticals Radiopharmaceutical Process 131 I-Hippuran F, ks, kpw "Tc-DTPA f, kpw 99mTc -DMSA 99m Tc-Glucoheptanoic Acid 9 9 m T c - M A G B F ,F , K

s ,kDw

Glomerular Filtration Rate The ideal glomerular filtration agent would have no excretion other than through glomerular capillary filtration. This tracer should also have no biologic metabolism and no plasma protein or cellular binding. These properties would allow the measurement of total body clearance of the tracer by simply measuring serial plasma activities in a gamma counter. The radiopharmaceutical 99mTc-DTPA is very close to such an ideal tracer.

After filtration by the glomerular capillary, the concentration of tracer in the proximal

convoluted tubule progressively increases as tubular fluid is reabsorbed. Before the tracer has a chance to enter the collecting system, the tubular activity will appear on a dynamic renal scintigram as progressively increasing renal cortical activity. The rate of renal cortical activity accumulation during the early portion of the study will reflect the rate of glomerular filtration. Therefore absolute quantitation of the glomerular filtration rate (GFR) is possible from: 1) an in vitro method with plasma sampling, 2) a non-invasive camera based method, or 3) a combined camera-based and limited plasma-sampling protocol. Many methods have been reported and compared in the literature (Fawdry et al., 1985; Ginjaume et al, 1986; Russell et al., 1985; Waller et al., 1987; Mulligan et al., 1990; Ham & Piepsz., 1991). Analytic methods which include plasma sampling are more accurate. The challenges encountered by past investigators included difficulty obtaining the plasma input function because the cardiac blood pool is typically outside of the renal imaging field and difficulty in quantitating renal activity because of attenuation. A variety of techniques have been used to try to make quantitative renal scintigraphy more accurate. Formulas of renal depth based on X-ray computed tomography measurements have been developed (Hindie et al., 1999; Inoue et al., 2000). One published report used a dual detector gamma camera and the geometric

RADIOPHARMACEUTICALS FOR THE STUDY OF LIVER AND RENAL FUNCTION

813

mean to compensate for attenuation and yielded relatively accurate results (Delpassand et al,, 2000). Accurate measurement of radiotracer activity in the abdominal aorta requires correction for both attenuation and partial volume effects, which are more easily performed with positron emission tomography (Germano et al., 1992). A clinically important application of

99m

Tc-DTPA renography is in the detection of renovascular hyper-

tension. On the baseline scan, no abnormalities in the dynamic scintigram or renogram may be seen because the kidney with renal artery stenosis will maintain a normal filtration rate by vasoconstricting the efferent arterioi and thereby increasing glomerular capillary pressure. After administration of captopril, there is loss of the efferent arterioi vasoconstriction leading to a decrease in glomerular capillary pressure, which then results in a decrease in glomerular filtration rate. The decrease in GFR will then appear on the scintigram as: 1) a delay in cortical tracer uptake, 2) a decrease in relative renal cortical activity (when compared to the non-stenosis side) and 3) a delay in the cortical washout.

Theoretically, the use of a parenchymal mean

transit time and an objective index should facilitate the detection of renal artery disease; however, this has not been found to be any better than more conventional analyses (Fine et at., 2000). A tubular secreted agent can also be used to detect the decrease in GFR induced by captopril in renovascular hypertension. Since the parenchymal (cortical) washout of a tracer residing in the proximal tubules is dependent on the GFR, a tubular secreted agent will also show a delay in washout after its peak activity (typically this washout period is within 3 to 30 minutes post injection). Using this principle, a variety of numerical indices has been shown to detect renovascular hypertension, such as the cortical-retention ratio (Sfakianakis & Bourgoingnie, 1987). In this study, all kidneys with significant renal vascular hypertension had either cortical retention of activity at 20 minutes in excess of 30% of the peak cortical activity or simply did not visualize. Excretion Index In acuce tubular necrosis (ATN), there is sloughing of the tubular cells and blockage of the tubular lumen, resulting in a drop in GFR; however, in milder forms of ATN, ERPF is only partially decreased. With a

814

HANDBOOK OF RADIOPHARMACEUTICALS

Figure 5. The use of two orthogonal parameters of renal function, excretion index (El) and effective renal plasma flow (ERPF), permits the diagnosis of a patient's kidney disease (Dubovsky, 1985; with author's permission).

tubular-secreted agent there should be prompt cortical uptake (relatively preserved ERPF); however, there will be a delay in cortical washout (decreased GFR). This decrease in GFR can also be conceptualized as the excretion index (El), which is the amount of radiotracer excreted from the body divided by the theoretical amount expected to be excreted based on the patient's measured ERPF (Tauxe, 1985). In chronic rejection, there is also a mild decrease in blood flow (ERPF); however, there is a relatively preserved GFR or excretion index. One can see that without the El, the ERPF alone is not able to differentiate between acute tubular necrosis and rejection (Figure 5). The ability of a tubular secreted tracer to be sensitive to a second physiologic process (GFR), in addition to its main radiopharmaceutical role (ERPF), permits us to classify this tracer as having the ability to measure orthogonal processes. A clinical application, using the orthogonal processes inherent in a tubular secreted agent, is in the evaluation of renal transplant function and its complications. The important clinical diagnoses extend from acute tubular necrosis to acute transplant rejection, chronic transplant rejection, and incomplete ureteral obstruction. The application of the excretion index (El) plotted versus the ERPF is being routinely performed on a clinical basis on renal transplant patients at the University of Alabama, Birmingham. The technique, which requires a single blood sample and urine samples, is considered by the U.A. clinicians as the most diagnostic of the renal function tests. They have performed this procedure in over 3000 cases with very reliable results (personal communication with Dr. E. Dubovsky).

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CONCLUSION Given the complexity of biological systems and the disorder imposed by disease, we should not be surprised that only two examples of orthogonality, hepatic scintiangiography and classification of renal function, could be found from almost fifty years of experience with liver and kidney radiopharmaceuticals. Although hepatic scintiangiography is rarely performed today, it forms the conceptual basis for hepatic CT angiography, which is a standard procedure for screening hepatic metastases. And although blood and urine sampling is tedious to perform, the calculation of El and ERPF has not been surpassed for over two decades. In conclusion, the radiologic techniques that produce two orthogonal parameters provide superior diagnostic power and have survived the test of time. We propose that future radiopharmaceutical be designed for multiple orthogonal parameters. Current strategies for the development of radiotracers, especially receptor-binding radiopharmaceuticals, employ a stage which includes a test of in vivo kinetic sensitivity to a physiologic or biochemical parameter. We propose that radiopharmaceutical design specifications include kinetic sensitivity to multiple processes. This strategy requires a strong multidisciplinary approach between radiochemist, data analyst, nuclear medicine physician, and clinician. The successful translation of chemistry to medicine can occur best when the radiochemist understands the physiologic and/or biochemical measurements that can be used by the clinician to diagnoses or manage a patient's disease. GLOSSARY A ABT ATN ASGP-R BSP CT CTC DTPA EC ECD El ERPF FDG GFR GSA h HBP HCC HIDA HSA HPLC ICG R15 IDA LDL

Angstrom (meter x 10-6) aminopyrine Breath Test acute tubular necrosis asialoglycoprotein receptor sulfobromophthalein computed tomography Child-Turcotte criteria diethylenetetraaminepentaacetic acid ethylene dicysteine ethylene cysteine dimer excretion index effective renal plasma flow fluorodeoxyglucose glomerular filtration rate DTPA-galactosyl human serum albumin hours hepatic binding protein hepatocellular carcinoma hepatobiliary iminodiacetic acid human serum albumin high performance liquid chromatography Indocyanine green retention at 15 minutes iminodiacetic acid low density lipoprotein

816 LHLl 5 MAGS MeV min NGA OIH PET PHMT PyG [R]0 RES Rmax ROC ROI SC SPECT TIPS

HANDBOOK OF RADIOPHARMACEUTICALS

99m

Tc-GSA

liver-to-heart plus liver ratio at 15 minutes mercaptoacetyl-triglycine mega-electron volts (106) minutes neoglycoalbumin ortho-iodohippuran positron emission tomography N-pyridoxyl-5-methyltryptophan pyridoxylidene glutamate receptor concentration reticulorendothelial system maximal removal rate receiver operating characteristic region-of-interest sulfur colloid single photon emission computed tomography transjugular intrahepatic portosystemic shunt

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Index Note: page numbers in italics refer to figures and tables a/p ratio 771 Ap42 protein 488 abscess, gallium-67 citrate detection 370 ABT-418 162 accelerators beam current 80 chemistry systems 80 choosing 79–81 energies 3, 76, 77, 80 isotope production 71–81 levels 71–2 linear 78–9 low energy 3 nuclear reactions 1 owning for biomedical research 81 particle 79-80 power 81 products 80 purchasing radionuclides 81 radio frequency 78–9 radionuclide production 1–54 shielding 81 tandem cascade 79 target number 80 vendors 80™1 ventilation 81 web sites 84–5 see also cyclotrons 3-acetamido-5-iodoproflavine, iodine125 labeled 784 acetanilidoiminodiacetate complex 332 acetate, carbon-11 labeled 532, 535 acetic acid fluorine-18 labeled 262 free radical scavenger 123 acetone, carbon-11 labeled 148, 247, 757, 759 acetophenone 267, 268 acetyl coenzyme A 151 carbon-11 labeled 153 acetyl hypofluorite, fluorine-18 labeled 251,313 acetyl-L-carnitine, carbon-11 labeled 153

acetyl-L-serotonin, carbon-11 labeled 153 acetylcholine 607 brain levels 609 dopaminergic modulation 617 GABAergic modulation 618 serotonergic modulation 617 acetylcholinesterase, activity estimation 514 acetylcholinesterase inhibitors, carbon11 labeled 163 acrylonitrile 149 acycloguanosine, fluorine-18 labeled 473–4 acycloguanosine analogs 471, 476–7 acyl chloride 148 acyl-CoA dehydrogenase deficiency 532 acylating agents, fluorine-18 labeled 261-2 addiction 557 nicotine 568 vulnerability 571-2 see also cocaine adenosine A] and A2 receptors 163 adenylate cyclase 583 adrenals, cocaine uptake 562, 563 adrenergic ligands 161–2 a-adrenergic receptors, myocardial 543 P-adrenergic receptors 755–9 CGP 12388 binding 756 density in lungs 755 ligands 755 myocardial 540–3 propranolol binding 583 see also beta-blockers airway smooth muscle, neural control 751 alanine, carbon-11 labeled 165 albumin carbon-11 labeled 168, 755 iodine-131 microaggregates 801 macroaggregated 331–2, 754 microaggregated 331, 801 aldehydes, carbon-11 labeled 156, 241

alkali metal acetate complex 251 alkali metals 27 alkenes 148, 199 carbon-11 labeled 247 fluorination 201 alkyl carbamates, carbon-11 labeled 242 alkyl halides 147 alkyl iodides, carbon-11 labeled 152. 243–4, 246 alkylating agents, fluorine-18 labeled 258–61 alkylthiols, carbon-11 labeled 245–6 alpha fetoprotein (AFP) gene therapy 468 alpha particle emitters 774–5, 781–3 linear energy transfer 781 survival curve 783, 784 therapeutic potential 781-3 alpha particles 767, 768, 771 cell killing 781 chromosomal aberrations 782 DNA double-strand breaks 782 linear energy transfer 772 radionuclide emission 774–5 alpha reaction on natural water 48 altanserin 609, 610, 617 fluorine-18 labeled 591, 593 altropane 518 alumina-based generator systems 110. Ill aluminium photomet ric detecti on 128 target body 50 targets 46 alveolar gas exchange 751 alveolar membrane 751 Alzheimer's disease amyloid plaque 344 cholinergic system 615 early diagnosis 738 5-HT2A receptors 593 serotonergic system 593 target specific radioligands 488–9 ubiquination abnormalities 588

824 amides carbon-11 labeled 156 solid metallic target irradiation 236 amines, nitrogen-13 labeled 125–6 D-amino acid peptides 694, 695 amino acids 644 aromatic 203–5 carbon-11 labeled 152, 164–6 endogenous 164–6 iodination 430 metabolism in brain/tumors 245 nitrogen-13 labeled 123–5 reactivity within protein 689 y-aminobutyric acid (GABA) see GABA aminodecane, nitrogen-13 labeled 125, 126 aminohexane, nitrogen-13 labeled 125 aminooctane, nitrogen-13 labeled 125 aminopolyether 256 ammonia 52 cryogenic solid targets 236 nitrogen-13 labeled 119, 120, 122-3, 125, 126 myocardial perfusion studies 530–1 preparation 754 quality assurance methods 127–8 renal perfusion 811 ammonium, quaternary resins 256 ammonium persulfate 430 amphetamine 586 ^-amphetamine 607-8 amygdala 586 amygdaloid nucleus 585 amyloid plaque 488 agents 344-5 (J-amyloid precursor protein (0-APP) 488 amyotrophic lateral sclerosis 588 androgen receptors 346, 719 bromine isotopes 452 prostate cancer 736–7 androgens fluorine substitution 727, 725 halofluorination 727, 725 iodine substitution 722-3 technetium-labeled pendant radiopharmaceuticals 732–3 angiogenesis a v pj integrin 671 markers 669–70 RGD sequences 671–2 angiotensin converting enzyme (ACE) inhibitor 761 annexin V 336 anti-epidermal growth factor receptor variant III (EGFRvIH) MAb 693, 694 anti-tenascin MAb 81C6 radioiodination 692 anti-tumor antibodies 426 antibiotics carbon-11 labeled 168 fluorine-18 labeled 210–11 antibodies antigenic target 685

INDEX iodination 433 labeling 690 properties 686–9 radiolabeled for tumor imaging/ therapy 685–705 see also monoclonal antibodies antidepressant drugs 160, 163 monoamine reuptake site binding 586 tricyclic 163 antiestrogens 715 antigen recognition site 688 antigen-antibody complexes 686 antigens, target 685, 686 antiogensin-II receptors 163 antipsychotic drugs atypical 590 dosage assessment 594 potency 594 antisense oligodeoxynucleotide, radiolabeled (RASON) 469, 478, 479 antitumor drugs nitrogen-13 labeled 126–7 platinum-195m 106 anxiety 590 apolipoprotein E (apoE) gene 488 apomorphine 585 apoptosis detection 336 argon gas 27 aromatase activity 164 aromatase inhibitors 715 aromatic aldehydes 148 aromatic compounds, fluorine-18 labeled no-carrier-added 262 aromatic nucleophilic substitution 199–200 aromatic ring substitution reaction 430 arsenic 26, 87 arsenic-72 25 arsenic-74 25 arthritis treatment 777 aryl fluorides 251 aryl halides 156 aryl-lithium compounds 251 fluorine-18 labeled 266 aryl-magnesium compounds fluorine-18 labeled 266 aryl-magnesium compounds, fluorine18 labeled 266 aryl rings, transition-mediated carbon11 cyanation 237 aryl inflates 156 aryl-zinc compounds, fluorine-18 labeled 266 Ascarite® trap 121, 235 asialoglycoprotein (ASGP) receptor 803, 804 analogs 804 asparagine, nitrogen-13 labeled 125 asparagine synthetase 124 L-aspartate 151 astatine-211 23–4 linear energy transfer 695, 772 monoclonal antibody labeling 6956,783 radiocolloid labeling 777

tellurium colloid 783 therapeutic potential 781-3 asthma 751 P-adrenoceptor density 756 muscarinic receptors 760 atenolol, carbon-11 labeling 541 atoms excess energy transfer 42 excitation 88 ionization 88 nuclear transformation 42 attention deficit hyperactivity disorder (ADHD) 564, 566 DAT as candidate gene 587 auditory cortex, FDG accumulation 584 Auger effects 767 Auger electron emitters 771, 783–6, 787 Auger electrons 106 bromine 441-2 5-bromo-2-deoxyuridine 450 cascade 446 extranuclear 769 indium-111 378, 389, 647 krypton 446 linear energy transfer 772 radionuclide emission 775–6 survival curves 768 automated systems 283–98 blood sampling 296, 297 butanol 287-8 diagnostics sensor data 293–4 documentation sensor data 293–4 engineering design 283 I8 FDG production 315 feedback control 290 fixed-plumbing 289 flow counting 296 hard-wired 285 input functions 296 liquid transfers 291, 292-3 loop method 297 microcomputers 286–7 microprocessor-based 286 modern synthesizers 294–6 physical parameter control 286 plasma analysis 296, 297 PLC 288 programming language 289 radiotracer synthesis 284 robot-controlled 287, 289, 295–7 solid phase extraction techniques 298 solvent evaporations 291-2 synthesis optimization 285 troubleshooting capabilities 293–4 unit operations design 288-9 real-time control 290–3 avidin 780 1 -aza-5-stannabicyclo[3.3.3]undecane 155 aziridines 166 azo dyes, technetium labeling 344, 345

825

INDEX b5 reductase 636 backing material 6 Baeyer-Villiger reaction 263 Balz-Sehiemann reaction 726 basal ganglia dopamine levels in Parkinson's disease 586 dopamine secreting neurons 585 BAT 412, 413 BCNC 127 BCNU 127 BD 1008 345, 346 behavior, motivated 611 benperidol, fluorine-18 labeled 208-9 benzamides 477 1,4-benzenedimethanol bimesylate 266 benzodiazepine receptors 125 bromine-76 labeled 454–5 flumenazil carbon-11 labeled 606 myocardial 544–5 radioligands 261 valium binding 583 S-benzoyl-mercaptoacetyltriglycerine (MAG3) see mereaptoacetyltriglycine (MAG3) benztropine, carbon-11 labeled 515, 606 chohnergic activity modulation 615– 16 dopaminergic effects on binding 617 vigabatrin effects on binding 618 benzyl alcohols, fluorine-18 labeled 263 benzyl halides, fluorine-18 labeled 263 beryllium 87, 95 beta-blockers 541–3 beta particle emitters 778–80, 781 survival curve 783, 784 beta particles 769, 771 high energy 770 linear energy transfer 771-2 long-range emission 773 radionuclide therapy 772–4 shorl-range emission 773 bile canaliculi 798 biliary excretion 801-3 bilirubin 798, 802 binding potential 506, 507–8 reversible ligands 508 bioelectrical impedance measurement 448-9 biomolecule iodination 433 biotin, yttrium-90 labeled 780, 757 Bis-Grignard reagents 157 bismuth 24, 87 bifunctional chela tors 704 bisrnuth-209(a,2n)astatine-211 reaction 696 bismuth-212 695 monoclonal antibody labeling 704, 783 therapeutic potential 781, 752 bismuth-213 695 linear energy transfer 772 monoclonal antibody labeling 704 therapeutic potential 781

bistosyloxyalkanes 259 bitosylate 266 bladder cancer, copper-67 labeled monoclonal antibody 413 bleomycin 30 blood cells indium-111-DTPA labeled 364 red 324 see also white blood cells blood flow, copper-labeled agents 406, 407-9, 410 blood sampling, automated 296, 297 blood volume estimation 448 imaging 324 blood-brain barrier transport 488 BMS-181321 537 BMS-194796 537 Bolton—Hunter reagent 427 bombesin 660–2 analogs 661–2 iodine-125 metaiodobenzoate labeling 660 bombesin receptor antagonists 660 bone agents 324, 325, 333 bone disease, osteolytic 664 bone marrow agents 331, 333 bone pain palliation 36 radiopharmaceuticals 773 radiostrontium 777 tin-117 106, 107 bone tumor localization with gallium 363 boron 52 boron-10 234 boron oxide 50–1 Bowman's capsule 807, 809 brachytherapy 426 brain 3-dimensional functional images 584 acetylcholine levels 609 addiction as disease 557 amino acid metabolism 245 chemical changes 557-8 chemical effects on mental activity 582 cocaine abuse 566–8 cocaine pharmacokinetics 560–2 dopamine levels 583 dopamine receptors 584–6 dopaminergic system 584–9 drug effects 582–3 FDG uptake 629 functionally activated regions 583 glucose metabolism 307, 310, 311 cocaine abuse 567-8 gray matter 629 imaging 307, 308 mental activity 584 monoamine oxidase 568–71 receptors 581, 582 brain agents, technetium-99m labeled 325-7 breast cancer 337 calcitonin uptake 666 diagnostic FDG-PET 631, 632

estrogen blocking 715 estrogen receptor-positive 207 estrogen receptors 346 imaging 735-6 FDG uptake 629, 630 iodine transporter 777 nodal involvement staging with FDG-PET 633 progesterone receptors 737 prognostic factors 735 response to treatment evaluation 634 bromide 448-9 biodistribution 443 bioelectrical impedance measurement 448-9 blood volume estimation 448 excretion 443 extracellular space estimation 448-9 bromine 25, 441-57 Auger electrons 441-2 2a-bromo-5a-dihydrotestosterone labeling 452 p-bromospireoperidol labeling 455 chemical labeling 446–8 decay-induced labeling 446 demetallation 447 dopamine receptor labeling 455–6 dry distillation 445–6 electrophilic reactions 446–7 enzymatic labeling 448 exchange reaction with radioiodine 429 fatty acid labeling 446, 449 labeling methods 446–8 macromolecule labeling 456 monoclonal antibody labeling 456–7 nucleophilic displacement 447–8 oxidation 446 peptide labeling 456 radioisotopes 721–4 half-lives 441, 442 production 444–5 properties 441–4 separation methods 445–6 radiopharmaceuticals 448–57 receptor site directed compounds 452-6 specific activity 448 bromine-75 12–14, 441, 442 production 444 reactions 12–13 radioisotope separation 13–14 targetry 13 bromine-76 14–15, 441, 442 benzodiazepine receptor labeling 454–5 bromine-79 ratio 448 5-bromo-2-thiouracil labeling 449 5-bromo-3-[[2(S)-azetidinyl]methoxy] pyridine labeling 453–4 5-bromo-6-nitroquipazine labeling 454 weta-bromobenzylguanidine labeling 453 4-bromodexetimide labeling 454 4-bromolevetimide labeling 454

826 bromine-76 (contd) bromolisuride labeling 455 (E)-N-(bromoprop-2-enyl)-2p-carbomethoxy-3p-4'-tolyl-nortropane labeling 456 bromospiperone labeling 455 2 p-carbomethoxy-3 -(4-bromophenyl)tropane labeling 455 dopamine DI receptor investigations 456 dopamine D2 receptor investigations 455 epidermal growth factor labeling 456 FBAU 452 FLB 457 labeling 455 monoclonal antibody labeling 457 muscarinic cholinergic receptor investigations 454 nicotinic acetylcholine receptor investigations 453–4 norepinephrine receptor investigations 453 PET imaging 443–4 positron emission 724 production 444–5 reactions 14 radioisotope separations 15 radiotracer 44 serotonin transporter labeling 454 jV-succinimidyl bromobenzoate labeling 456 targetry 14 UdR 450–1 bromine-77 15–17, 441, 442, 445 androgen receptor investigations 452 16a-bromo-11 p*-methoxyestradiol17P labeling 452 16a-bromoestradiol-17oc labeling 452 17-bromoheptadecanoic acid labeling 449 a-bromostearic acid labeling 449 clonal survival 784 estrogen receptor investigations 452-3 fibrinogen labeling 449 hallucinogen receptor investigations 453 labeling 446 production reactions 15 radioisotope separation 17 spallation production 445 targetry 16–17 UdR 442, 450–1 bromine-79:bromine-76 ratio 448 bromine-80 441,442 production 444 bromine-80m 446 estrogen-directed therapy 453 non-steroidal estrogen labeling 452 production 444 bromine-82 441,442 FBAU 451 GABAA receptor channel opening 449 production 444

INDEX bromine-carbon bond 443 5-bromo-2-deoxyuridine (BrUdR) 442, 450–1, 453 5-bromo-2-thiouracil 449 5-bromo-2'fluoro-2'-deoxyuridine (FBAU) 451-2 5-bromo-3-[[2(S)-azetidinyl]methoxy] pyridine (BAP), bromine-76 labeled 453–4 2a-bromo-5a-dihydrotestosterone, bromine labeled 452 5-bromo-6-nitroquipazine, bromine-76 labeled 454 16a-bromo-l 1 fJ-methoxyestradiol-l 7p", bromine-77 labeled 452 bromoantipyrene 453 mefa-bromobenzylguanidine (MBBG), bromine-76 labeled 453 bromocryptine 586 4-bromodexetimide (BrDEX), bromine-76 labeled 454 16a-bromoestradiol-17a, bromine-77 labeled 452 bromofluorination 727, 725 17-bromoheptadecanoic acid, bromine77 labeled 449 4-bromolevetimide (BrLEV), bromine76 labeled 454 bromolisuride, bromine-76 labeled 455 bromoperoxidase 448 2-(6-(4-bromophenoxy)hexyI)oxirane2-carboxylic acid 449 (E)-N-(bromoprop-2-enyl)-2p-carbomethoxy-3p-4'-tolyl-nortropane (PE2Br), bromine-76 labeled 456 3-bromopropyl-l-triflate 261 bromospiperone, bromine-76 labeled 455 />-bromospireoperidol (BrSP), bromine labeled 455 a-bromostearic acid, bromine-77 labeled 449 bromosulfophthalein 801–2 2-(5-bromothienyl)-2-thienylglycolate (BrQNT) 454 bromovinylestrogens, steroidal 453 BRU-59-2 330–1 BRU59-21 537 BSM-181321 330 Bucherer-Strecker synthesis 165 bunazosin, carbon-11 labeled 543 busulfan 159 butanol automated synthesis 287-8 oxygen-15 labeled 131 butyl iodide, carbon-11 labeled 243, 244 butyrophenone neuroleptics 477 fluorine-18 labeled 208-9 C-2 308–9, 310, 311 C-C bond formations 153 C-metal bond 447 c-myc oncogene 478 C18 Sep-Pak® 298 CA125 385 cadmium 39, 40

calcitonin 664–6 analogs 665–6 receptors 664, 665 calcium channel antagonists 163 calcium phytate 331 cancer diagnosis 392-3 hormone-responsive tumor receptor imaging 715–39 nuclear hormone receptor imaging 737–8 sigma receptors 715–16, 720, 723–4 carbon-11 labeling 731 fluorine-substituted ligands 727–8. 730 imaging 738 technetium-labeled 734 cancer chemotherapy 211 gallium-67 determination of response 370 see also multidrug resistance cancer treatment bromine isotopes 441 carrier molecules 771 hormone therapy 715 pretargeting 705 radionuclide selection 767–87 captopril 813 fluorine-18 labeling 761 carazolol, carbon- II labeled 541, 758–9 carbamoyl compounds, carbon-11 labeled 157 carbohydrates carbon-II labeled 166–8 metabolism in liver 797 synthesis 153 2p>-carbomethoxy-3pX4-bromophenyl)tropane (fJ-CBT), bromine-76 labeled 455 carbon 762 cold carrier 44 technetium-99m labeled particles 754 carbon-II 42, 48–51 acetate labeling 532, 535 acetone labeling 148, 247, 757, 759 acetyl coenzyme A labeling 153 acetyl-L-carnitine labeling 153 acetyl-L-serotonin labeling 153 acetylcholinesterase inhibitor labeling 163 alkene labeling 247 alkyl carbamate labeling 242 aIkyl iodide labeling 152, 243–4, 246 alkylthiol labeling 245–6 alpha-receptor labeling 543 amide labeling 156 amino acid labeling 152, 164–6 antibiotic labeling 168 applications 141–2 automatic synthesis 286 benztropine labeling 515, 606 cholinergic activity modulation 615–16 dopaminergic effects on binding 617

827

INDEX vigabatrin effects on binding 618 beta-blocker labeling 541–2 biosynthesis 143 carazolol labeling 541, 758–9 carbohydrate labeling 166–8 carbon dioxide labeling 145–6, 148 carbon monoxide labeling 150, 154, 155–6 carboxylic acid labeling ! 57 CFT labeling 519-20 cocaine labeling 559, 560–2, 565 cyanamide labeling 145, 149 cyanate labeling 149 cyanide labeling 148, 235-8 cyanogen bromide labeling 149-50, 168, 238 cyanopropanol labeling 159 DACA labeling 168 t-deprenyl labeling 505, 516 dihydrotetrabenazine labeling 517, 519 1-3,3-dimethylheptadecanoic acid labeling 532 DOPA labeling 164 dopatnine labeling 166, 244 enantiomers 145 endogenous compound labeling 164–8 enzymes catalysis 143 labeling synthesis 151–3 ephedrine labeling 161–2 epinephrine labeling 153, 166, 538–9 erythromycin labeling 168, 762 estrogen labeling 730, 731 ethanethiol labeling 246 ethanol labeling 148 ethyl iodide labeling 147, 243, 244 fatty acid labeling 167 FLB 457 labeled 297 fiumenazil labeling 125, 295–6, 606 foil material on targets 50 formaldehyde labeling 147-8, 153, 168, 241–2 formoterol labeling 542, 759 o-fructose labeling 153 GB67 labeling 161, 543 glucose labeling 153, 167, 531 granisetron labeling 161 guanidine labeling 145 half-life 142 halonitrile labeling 148, 238 hydrogen cyanide labeling 145, 148, 149 5-hydroxy-L-tryptophan labeling 164 4-imidazolyl group labeling 159 iodomethane labeled 297 isotopic dilution 144 isropidine labeling 163 ketamine labeling 163 ketone labeling 156 kinetics 143 labeled compounds 141–69 labeled precursors 145–50 labeling position 144

labeling strategies 142-5 lithium methyl(2-thienyl)cuprate labeling 157 macroaggregate labeling 755 McN5652 labeling 591, 592 melatonin labeling 166 mesterolone labeling 157, 167 metahydroxyephidrine labeling 53840 metaraminol labeling 161, 538, 539 methane labeling 145, 146, 148 methane sulfonyl chloride labeling 147 methanethiol labeling 246 methanol labeling 148 methionine labeling 153, 165, 245, 634 methyl bromide labeling 147 jV-methyl-D-aspartate (NMDA) receptor complex ligand labeling 162–3 methyl-labeled albumin 755 methyl lithium labeling 147, 240, 247, 730 N-methyl-piperidin-4-yl-2-cyclohexyl2-hydroxy-2-phenylacetate labeling 760–1 methyl triflate labeling 146–7, 240–1, 297 methylating agent labeling 239–41 methylene iodide labeling 145 ,146–7 1-p-methylheptadecanoic acid labeling 532 methylhypofluorite labeling 147 methylisocyanate labeling 148 methylphenidate labeling 517, 519, 559, 565 W-methylspiperone labeling 297, 584, 587, 591, 594 A^methylspiroperidol labeling 514, 605 microaggregate labeling 755 MQNB labeling 544 neuroreceptor ligand labeling 729 nisoldipine labeling 163 nitroethane labeling 245 nitrogen gas production 44 nitromethane labeling 244, 245 nitropropane labeling 245 nitrosourea labeling 168 nitrostyrene labeling 244 NNC 112 labeling 517 norepinephrine labeling 167, 245 norphenylephrine labeling 245 nuclear reactions for production 232-3 nucleogenic atoms 231–2 octopamine labeling 167 opioid receptor ligand labeling 162 organic synthesis 143 ornithine labeling 165 oxide labeling 233-5 oxygen labeling 233-5, 247 ozone labeling 243 1-palmitate labeling 532 w-palmitate labeling 532-3

peptides labeling 166 PET 141–2, 159–64 PET labeled precursor production 232–48 phenethylamine labeling 244 phenylalanine labeling 152, 165 phenylephrine labeling 162, 53840 phosgene labeling 150, 161, 242–3, 246–7, 756 phosphonium salt labeling 247-8 PK11195 labeling 544 position-specific labeling 143 positron emission 48-9 precursor labeling 232-48 primary precursors 145-6 procaterol labeling 247 production 76 reactions 48-9 progesterone labeling 157, 167 propanethiol labeling 246 propyl iodide labeling 243, 244 propyl ketene labeled 148 prostaglandin labeling 155 quinoxaline labeling 158 radioisotope separation 50–1 receptor-binding radiopharmaceutical labeling 729–31 recoil labeling 143 remotely controlled synthesis 285 ring closures 158-9 Ro 15-1788 labeling 297 Rolipram labeling 297 rotenoid labeling 164 roxithromycine labeling 168 SarCNU labeling 168 Schering 23390 labeling 297, 517 secondary precursors 146–50 selenomethionine labeling 165 semotiadil labeling 163 serine labeling 153 serotonin labeling 762 specific activity 43, 44 specific radioactivity 144 steroid labeling 167–8 substitution onto biomolecules 42 synthesis 143 methods 145-57 remotely controlled 285 tamoxifen labeling 168 targetry 49–50 technical approaches 142–3 thiocyanate labeling 149 thymidine labeling 635 tracers 141–2 biological targets 144–5 production 142–4 transition metal-mediated reactions 153-7 trifluoracetonitrile labeling 149 tropanyl benzilate labeling 515, 606 tyramine labeling 167 urea labeling 148, 242, 246–7 water labeling 241 WAY 100635 labeling 517–18, 590, 593

828 carbon-11 (contd) see also methyl iodide, carbon-11 labeled; phosgene, carbon-11 labeled; raclopride, carbon-11 labeled carbon-12 43, 87 carbon-12(d,n)nitrogen-13 reaction 754 carbon-13 51 carbon-13(p,n)nitrogen-13 reaction 754 carbon-14 87, 88 decay 778 deoxyglucose labeling 495–6, 583 18 FDG labeling 311 8-ganciclovir labeling 474 carbon dioxide 48, 49, 50 carbon-11 labeled 145–6, 148 secondary precursors 146 exchange 751–62 frozen oxygen-enriched targets 48 labeling 52 nitrogen-13 production 754 oxygen-15 labeled 131 oxygen-18 enriched 255 carbon monoxide carbon-11 labeled 150, 154, 155–6 labeling 52 oxygen-15 labeled 131 carbon monoxide-erythrocytes, carbon-11 labeling 762 carbonyl fluoride 251 carbonyl functionality 150 carbonylations 155–7 carboxylic acid salts 148 carboxylic acids, carbon-11 labeled 157 2|}-carboxymethoxy-3p(4[18F]fluorophenyl)tropane 209 carcinoembryonic antigen (CEA) 385, 685 technetium-99m labeled 701–2 cardiac dysfunction 529 see also heart cardiac ejection fraction, technetiumlabeled red blood cells 324 cardiac imaging, fluorometaraminol 206 cardiomyopathies 449 carfentanil 162 carrier molecules 771 catecholamines fluorine-18 labeled 558 positron-labeled derivatives 538–40 caudate nucleus 585, 586 CCNU 126 CD33 cell surface antigen 704 CEA-Scan 701–2 cell proliferation 168 cell radiodensity 770 cellular responses to radionuclides 768-70 cerebellum raclopride Bmax/Kd' 512 serotonergic innervation 589 tracer distribution/binding models 503–4 cerebral blood perfusion 90, 407

INDEX copper-62-PTSM 408 regional 325, 327 cerebral cortex, serotonergic innervation 589 cesium fluoride 251 C-Fbond 309 CFT, carbon-lllabeled 519–20 (S)-CGP 12177 755–6 (S)-CGP 12388 756–7 CGP 20712A 543, 759 carbon-11 labeled 161 CGP12177, carbon-11 labeled 161, 541, 542 CGP 12388, carbon-11 labeled 541, 542 chelating agents, bifunctional (BFCs) 404, 698-9, 704, 705 chloramine-T 427, 430, 431, 432, 449 bromine-76 labeled compounds 454 co-chlorination of substrate 447 monoclonal antibody labeling 690 chlordiazepoxide 449 l,3-bis(2-chlorethyl)-l-[13N]nitrosourea see BCNU l-(2-chlorethyl)-3-cyclohexyl-3-cyclohexyl-3-cyclohexy-1 -nitrosourea see CCNU chlorination, catalytic 243 chlorine 27 ultraviolet photochemical reaction 242 chlorine-34m 11–12 production reactions 11 radioisotope separation 12 targetry 12 jV-chloro compounds 447 jV-chloro-iodotyramine 427 chloroperoxidase 448 jV-chlorosuccinimide 447, 451, 691 monoclonal antibody labeling 690 W-chlorotoluenesulfonamide 447 chlorpromazine, carbon-11 uptake 762 cholecystokinin (CCK) 667–9 indium-Ill DOTA labeling 669 cholecystokinin B (CCK-B)/gastrin receptor 667-9 cholescintigraphy 332 cholesterol synthesis 798 choline acetyltransferase 608 cholinergic activity modulation with benztropine 615–16 cholinergic ligands 162 cholinergic receptors 608 chromate phosphate colloid, phosphorus-32 labeled 799, 800 chromatidal aberrations 768 chromic acid 430 chromium 28 catalyst 237 chromosomal aberrations 768, 782 chronic obstructive pulmonary disease 751, 762 muscarinic receptors 760 chrysamine G 344, 345 chylomicrons 797 cimetidine 451, 452, 583 cingulate cortex 586

cingulate gyrus cocaine abuse 568 serotonergic innervation 589 ciprofloxacin, technetium labeled 339 cisplatin 106 nitrogen-13 labeled 127 cisternography, indium-111-DTPA 364 P-CIT 160, 591 iodine-23 labeled 592-3, 594 citalopram 160, 610 clorgyline 163 closed loops 142 clozapine 590, 594 cobalt-55 29–31 production reactions 30 radioisotope separation 30–1 targetry 30 cobalt-56 30 cobalt therapy, remotely controlled 426 cocaine 160 abuse 611 analogs 209 behavioral high 561,562 brain imaging of abusers 566–8 brain pharmacokinetics 560–2 carbon-11 labeled 559, 560–2, 565 cardiotoxicity 562–3 cholinergic activity modulation 616 clearance rate 565 dopamine D2 receptors 568, 572 dopamine function in brain 566–8 dopamine terminal uptake 561, 562 dopamine transporter blockade 560. 561-2, 565 enantiomers 561–2 hydrogen-3 labeled 560 metabolism 560, 567 methylphenidate comparison 564–5 peripheral organ distribution/kinetics 562–4 PET studies 560–8 pharmacokinetics 565 psychostimulant effects 586 raclopride studies 568 receptor binding 586 time-activity curve in brain 566 colchicine, carbon-11 labeled 168 colon carcinoma diagnostic FDG-PET 632 somatostatin receptors 646 yttrium-90 labeled DOTA-biotin 701 colony-forming assay 768 colorectal cancer 337 carcinoembryonic antigen 385 copper-67 labeled monoclonal antibody 413 I8 FDG in diagnosis/staging 392 recurrence diagnosis 365, 634 complementarity determining region (CDR) 688 compound nucleus model 1-2, 90 computed tomography (CT), liver tumors 801

829

INDEX conductivity sensors 293 congo red 344, 345 copper 34, 35–6, 401–16 arsenic target 26 biochemistry 405–7 biomolecule labeling 404 blood flow agent labeling 406, 4079,410 chelators 404–5 bifunctional 699 chemistry 403–4 coupling reactions 157 DTS complexes 411 hypoxia agent labeling 410–12 monoclonal antibody labeling 41214 peptide labeling 414–15 photometric detection 128 positron-emitting radionuclides 402 protein labeling 412–14 PTSM 406, 407, 410 radioisotopes 31–3 separation at trace concentrations 112 copper(l) 403–4 salts 237 copper(II) 403, 404 bis(thiosemicarbazone) complexes (Cu-BTS) 401–9,410 charge 405 thiosemicarbazones 404, 406, 407, 410, 411 copper-60 401 production 402, 403 copper-61 31, 32, 401 production 402, 403 copper-62 32, 401 BTS 401-9,410 ETS 407, 409 production 402, 403 n-PrTS 407,409 PTSM 408–9, 530 copper-64 31, 32–3, 401 BAT-2IT-1A3 413 F(ab')2 fragments 414 monoclonal antibody labeling 41214, 699 octreotide labeling 651 production 402 targeted radiotherapy 644 TETA-octreotide 414, 415 copper-67 401 cost 689 F(ab) 2 fragments 414 monoclonal antibody labeling 41214, 699 nuclear reactor production 111–12 production 402, 403 copper-bis(thiosemicarbazone) (CuBTS) 406, 407 hypoxia imaging 410, 411 copper-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) 411 corpus striatum 607 corticosteroids, fluorine-18 labeled 738 cosmic rays 87

Coster-Konig electrons 775 Coulomb barrier 3, 90, 91 Coulomb forces 88 Crohn's disease 339 somatostatin receptors 645 cross-couplings 154–5 cross-section 91, 92 cyanamide, carbon-11 labeled 145, 149 cyanates, carbon-11 labeled 149 cyanations 154 cyanide carbon-11 labeled 148, 235–8 non-synthetic preparation 236–8 synthetic preparation 235–6 recoil labeling 236 solid metallic target irradiation 236 transition-mediated carbon-11 cyanation of aryl rings 237 cyanogen bromide, carbon-11 labeled 149-50, 168, 238 cyanomethyl pivalate 149 cyanopropanol, carbon-11 labeled 159 /ra/is-cyclohexyldiethylenetriamine pentaacetic acid (CHX-A-DTPA) 704 cyclosporin A 348, 349 cyclotrons 71-3 beam loss 75 components 73, 74 deflection system 75 exit ports 75 extraction foil 75 extraction system 75 ion source 75 negative ion 73, 75 PET 73 principles 73–5 proton only 79–80 small low-energy 73 variable energy feature 75 web sites 84–5 see also accelerators cystic fibrosis 751 cytokines, tumor survival/proliferation 669 cytosine deaminase 470 D-KRYRR 694, 695 DACA, carbon-11 labeled 168 dees 73, 74 2-deoxy-2-[18F]fluoro-D-glucose see 18 FDG 2-deoxy-2-chloro-D-glucose (Cl DG) 316–17 2-deoxy-2-fluoro-D-mannose, fluorine18 labeling 313–14 3-deoxy-3-bromo-D-glucose 447 3 '-deoxy-3 '-fluorothymidine, fluorine18 labeled 635 2-deoxy-D-glucose 308–9, 310, 311 -2 for fluorine substitution 309, 310, 311 metabolism in brain 310 deoxyglucose, carbon-14 labeled 4956, 583 L-deprenyt 163

carbon-11 labeled 505, 516 smoking cessation 571 depreotide 654–5 depression 590 delusional 594 5-HTiA receptors 593–4 5-HT2A receptors 594 serotonergic system 593–4 serotonin transporter 594 smoking 569 desferrioxamine 650 desipramine 594 deuterons 79, 80 DeVarda's alloy 123, 128 dextrallophan 345 di-lithium tetrachlorocuprate 157 diaminedithiol (DADT) chelators 661 diaryliodonium salts 156 diazepam 125 6-diazo-5-oxy-L-norleucine (DON) 451-2 diazonium chloride 267 diazonium fluoride derivatives, aromatic 726 dibromodimethylhydantoin 727 m-dichlorodiamineplatinum (II) see cisplatin diethylenetriaminepentaacetic acid see DTPA dihalotriphenylphosphme 265 Sa-dihydrotestosterone 347 16p-5a-dihydrotestosterone, fluorine18 labeled 736–7 dihydrotetrabenazine (DTBZ), carbon11 labeled 517, 519 3,4-dihydroxy-L-phenylalanine see DOPA diiodomethane 258 diiodosilane 265 dilactitol-tyramine (DLT) 693 dimercaptosuccinic acid (DMSA) 334 R(-)-2,5-dimethoxy-4-77bromoarnphetamine(DOB) 453 dimethoxyphenylisopropylamine 446 dimethylammopyridinium 316 4-(dimethyIamino)pyridiniumbrornide perbrornide 268 dimethylgallium 735 1-3,3-dimethylheptadecanoic acid, carbon-11 labeled 532 Af,./v*-dimethylphenethylamine 163 Af-(2,6-dimethylphenyoicarbamoylrnethyl)-iminodiacetic acid (HIDA), technetium-99m labeled 325, 8023 Diodrast 426 diphenylamine 247 diphosphonates 333 diprenorphine 162, 261 disofenin, technetium-99m labeled 803 distribution volume (DV) 506, 507 generation from distribution volume ratio 512 graphical analysis method 518–19 model selection 516–17 plasma levels 510, 517

830 distribution volume (DV) (contd) radiotracers 605–6 ratio 506, 507–8 reference region 512 receptor 506, 507 reference region 506, 507 reversible ligands 508, 510 tissue to plasma ratio 510 underestimation 515 dithiaphosphine, tetradentate 661 division delay 769–70 DMP444 340 DMP757 339–40 DNA bromine-76-FBAU labeling 452 damage 767, 768, 785 double-strand breaks 767, 782 single-strand breaks 767 solid phase supported chemistry 284 DOPA 151 carbon-11 labeled 164 fluorine-18 labeled 204–5 L-DOPA 583, 586 dopamine acetylcholine effects 617 antagonists 583 brain 583 carbon-11 labeled 166, 244 cholinergic modulation 608–9 cocaine abuser brain function 566–8 elevation response to cocaine 560 endogenous 605 activity 608-9 fluorine-18 labeled 206, 540 GABAergic modulation 610–11 schizophrenia 612–15 substance abuse 611–12 interactions in extra-pyramidal motor system 607 nitrogen-13 labeled 125 Parkinson's disease 582-3, 586 reuptake blocking 564, 586 sites 586 serotonergic modulation 609–10 substantia nigra neurons 585 synaptic 586 transporter ADHD 564 cocaine binding 560, 561–2, 565–6 methylphenidate blocking 564–6 occupancy 565–6 studies 160 dopamine antagonists 159–60 dopamine D| binding site 261 W-methylbenperidol selectivity 208 dopamine D| receptors 159, 585 bromine-76 labeling 456 dopamine D2 binding site 208 dopamine D2 receptors 159, 230, 585 bromine-76 labeling 455 clozapine occupancy 594 cocaine abuse 568, 572 corpus striatum 607 dimerization 587

INDEX gene therapy monitoring 213 Af-methylspiperone binding 506 neuroleptic-treated patients 587 nicotine effect on occupancy 612 reporter gene imaging 477-8 schizophrenia 587 selective antagonists 212 variant 571-2 dopamine D3 receptor selective antagonists 212 dopamine 04 receptors 587 dopamine receptors binding 583 brain 584–6 bromine labeling 455–6 imaging 585 types 585 dopamine system in Lesch-Nyhan/ Tourette disease 589 dopamine transporter protein (DAT) 341–2, 586–7 distribution 586 gene 587 cloning 586 polymorphisms 587 Parkinson's disease 588, 589 dopaminergic activity modulation 607-8 dopaminergic ligands 159–60 dopaminergic neurons 608 inhibitory effect 585 post-synaptic 586 dopaminergic projections, mesocorticolimbic 611 dopaminergic system, brain 584–9 dose-response survival curves 768–9 DOTA copper labeled bifunctional chelators 404, 405 indium-Ill labeled MAb conjugation 703 yttrium-90 labeling of monoclonal antibodies 699 DOTA-biotin, yttrium-90 labeled 700-1 DOTA-J415, indium-Ill labeled 703 DOTA-J591, indium-Ill labeled 703 DOTA-lanreotide 654 double shoot method 79 drug abuse, DAT as candidate gene 587 drug abuse PET research 557-73 addiction vulnerability 571-2 mechanistic studies 559 smoking 568-71 tracer kinetics 559 see also cocaine drugs, pharmaceutical brain effects 582-3 development 581 DTPA analogs 699 copper(II) complexes of bifunctional chelators 404 coupling to MAb 694 indium-Ill labeled 364, 375, 382, 383, 390-1

structure 386 technetium-99m labeled 754, 808, 812, 813 yttrium-90 labeling of monoclonal antibodies 699 see also MyoStint; ProstaScint DTPA-7E11.C5.3 see ProstaScint DTPA-B72.3, indium-Ill labeled 702 DTPA-T3-TATE 415 dysprosium-166 108, 109 7E11 monoclonal antibody 703 EDTA analogs 699 copper(II) complexes of bifunctional chelators 404 EF5 201 effective renal plasma flow (ERPF) 811–12, 814 electro-deposition technique 112 electrons capture 775 energetic 767 energy 88 Electrospray mass spectrometry 448 endocrine tumor proliferation marker 451-2 endonorbornyl-p-tolylsulfonamide 255 endopeptidase tracers 163–4 endorphins 213 endothelial cells, otvpj integrin 670–1 endothelial receptor systems 751 endothelin receptors 163 energy excitation 2, 92 nuclear reactions 2, 3, 91, 94 threshold 91 enolmethyl ethers 147 enrichment factor 97 entorhinal cortex, dopamine D4 receptors 587 enzymes compounds for PET study 159–64 inhibitors 163–4 kinetic data 487 labeling synthesis 151–3 lung function control 751 substrates 163–4 ephedrine, carbon-11 labeled 161–2 epibatidine 162, 212 epichlorohydrine 148 epidermal growth factor, bromine labeled 456 epilepsy 610 epinephrine carbon-11 labeled 153, 166, 538–9 fluorine-18 labeled 540 ergocryptine 446 erythrocytes autologous 332 labeling 333 erythrocytes-carbon monoxide, carbon-11 labeling 762 erythromycin, carbon-11 labeled 168, 762

INDEX esophageal cancer staging with FDGPET 633 estradioi fluorine-18 labeled 207, 287 6-keto-2-trirnethylammonium analog 726, 727 technetium labeled 347 estrogen(s) carbon-11 labeling 730, 731 derivatives 446 fluorine-18 labeling 735 fluorine substitution 725-6, 727 gallium substitution 735 integrated ligands 733-4 organometallic systems 734 iodine-123 labeled 735 iodine substitution 722 technetium-labeled pendant radiopharmaceuticals 732–3 estrogen receptors 346, 582 imaging in breast cancer 735–6 ligand labeling 721–2 ethanethiol, carbon-11 labeled 246 ethanol carbon-11 labeled 148 free radical scavenger 123 ether, 18-crown-6 257 ethyl cysteinate dimer (BCD) 326, 327 ethy! iodide, carbon-11 labeled 147, 243, 244 exchange labeling 324 excitation energy 2, 92 excitation sources, external 232 extra-pyramidal motor system 607 extracellular space estimation bromide 448-9 F(ab')2 fragments 687, 688 Fab fragments 643, 687, 688 /ac[99mTc(CO)3(H2)3]+ 652 fallypride 212 fatty acids P-oxidation 532 bromine-labeled 446, 449 carbon-11 labeled 167 fluorine-18 labeled 533 heart imaging 529 co-iodo esters 157 iodophenyl 532–3 rnyocardial metabolism 531–5 PET tracers 532–3 SPECT tracers 534–5 radioiabeled 531–5 saturated 157 18 FDG 45, 307–17, 558 Alzheimer's disease 488–9 automatic synthesis 285, 287, 315 feedback control 290 brain glucose metabolism in cocaine abuse 567-8 breast tumor imaging 735 carbon-14 labeled 311 cyclotron facilities 409 cyclotron-produced 581 depression 593 design 308-9, 310, 311

distribution 629, 630 dynamic PET 520 electrophilic preparation 312, 313 glucose metabolism 629–31 granulation cell uptake 630 heart imaging 529 inflammatory cell uptake 630 isomerase enzyme inhibition 796 low body background 311 mannose isomer production 313–14 metabolism in brain 310 microcomputer controlled synthesis 285 rnyocardial glucose metabolism 531 no-carrier added form 315 positron emission 584 production 531 prostate cancer 7J7 radiation dosimetry 312 renal tubules 808 safety 312 structure 308 supply 307 synthesis animal studies 311-13 cation exchange resin 316 deprotection step 316, 317 developments 316–17 electrophilic route 311, 313, 314 human studies 311–13 Kryptofix 2.2,2. 287, 314–15, 316, 317

nucleophilic route 314, 316 nucleophilic substitution 287 synthetic routes 313 toxicity studies 312 uptake brain 567-8, 629 heart 529, 629 tumors 468, 630 uses 313, 392 yield 314 FDG-6-P 629 FDG-PET imaging 629–36 diagnosis 631, 632 oncological applications 631–6 recurrent disease diagnosis/staging 633–4 response to treatment evaluation 634 staging 632-3 fenfluramine 610 ferric sulfate 430 fibrinogen 449 Pick principle 811 fission 96 products 90 flavone synthesis 156–7 FLB 457 bromine-76 labeled 455 carbon-11 labeled 297 FLB 463, bromine-76 labeled 455 D-flenfluramine 593 fleroxacin, fluorine-18 labeled 210 flow counting automated systems 296 fluconazole, fluorine-18 labeled 211 flumenazil, carbon-11 labeled 125, 606

robot synthesis 295-6 fluoralkylating agents, fluorine-18 labeling 260 fluorescein isothiocyanate 427 fluoride-19(p,n)neon-19 reaction 753 fluoride ions 46–7 anhydrous 256 counterions 256 exchange reaction 253 fluorine-18 labeling 255-7 kryptofix 2.2,2. use 314–15, 316 reactivity 257 resin-supported 316 solubility maintenance 256 target water 256 high specific activity nucleophilic 200 nucleophilic aromatic substitution 726 fluorination electrophilic 200–1 fluorine-18 labeling of reagents 250-5 nucleophilic 197–200 nucleophilic reagents 250 fluorine-18 labeling 255–7 regioselective 201 fluorine, elemental 46 bromine comparison 443–4 fluorine-18 labeled 250-1, 311, 312, 313 fluorine-18 42, 44-8 acetic acid labeling 262 acetyl hypofluorite labeling 251, 313 activating groups 200 acycloguanosine labeling 473–4 acylating agent labeling 261–2 aliphatic nucleophilic displacements 198-9 alkylating agent labeling 258–61 altanserin labeling 591, 593 anhydrous source 250 antibiotic labeling 210–11 aqueous.source 250 aromatic nucleophilic substitution 199–200 aryl-lithium compound labeling 266 aryl-magnesium compound labeling 266 aryl-zinc compound labeling 266 benperidol labeling 208–9 benzyl alcohol labeling 263 benzyl halide labeling 263 beta-blocker labeling 542 bromine-76 PET comparison 443–4 captopril labeling 761 cation addition 197–8 chemistry 195–213 corticosteroid labeling 738 cyclotron production 249 decay 195 2-deoxy-2-fluoro-D-mannose labeling 313-14 3'-deoxy-3'-fluorothymidine labeling 635 deuteron reaction 79

INDEX

832 fluorine-18 (contd) 16p-5ot-dihydrotestosterone labeling 736–7 DOPA labeling 204–5 dopamine labeling 206, 540 electron-withdrawing groups 200 electrophilic fluorinating agent labeling 250–5 electrophilic fluorination 200–1 elimination from nitrogen-13 labeled ammonia 127 epinephrine labeling 540 estradiol labeling 207, 287 estrogen labeling 735 fatty acid labeling 533 FHBG production 475–6 FHPG production 475–6 fleroxacin labeling 210 fluconazole labeling 211 fluoralkylating agent labeling 260 fluorination methods 197–201 fluorine labeling 250–1,311, 512, 313 2-fluoro-2-deoxy-r>glucose labeling 252 l-fluoro-2-pyridone labeling 254 2-fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine labeling 212 fluoro-16ot-ethyl-19-norprogesterone labeling 208, 737 3-fluoro-o-methyltyrosine labeling 203–4 6-fluoro-L-dopa labeling 294–5 fluoro-L-m-tyrosine labeling 205 yV-fluoro-W-alkylsulfonamide labeling 255 fluoroacetone labeling 758 8-fluoroacyclovir labeled 474 fluoroacylating agent labeling 261–2 fluoroalanine labeling 263 fluoroalkane labeling 258, 259 fluoroalkyl halide labeling 259 fluoroalkylbenzylsulfonate ester labeling 266 2-fluoroaniline labeling 252 2-fluoroanisoIe labeling 252 fluoroaromatic labeling 201 fluoroaryl precursor labeling 262–8 .fluoroarylketone labeling 267–8 fluorobenzaldehyde labeling 263, 264 fluorobenzene-diazonium chloride labeling 267 4-fluorobenzoic acid labeling 262 fluorobenzyl bromide labeling 264 fluorobenzyl iodide labeling 264 fluorobenzylalcohol labeling 263–4 fluorobenzylhalide labeling 263, 264–6 fluorobenzyltrozamicol labeling 264 4-fluorobromobenzene labeling 267 4-cis-fluorocaptopril labeling 761 fluorocarazolol labeling 542, 757–8 fluorocarboxylic acid labeling 261 fluorocatechol labeling 263 fluoroCGP 12388 labeling 542 fluorochlorobenzene labeling 267

fluorodeprenyl labeling 263 6-fluoroDOPA labeling 204–5, 263, 558 fluorodopamine labeling 206, 540 fluoroestradiol labeling 287, 726, 727 16ot-fluoroestradiol labeling 207, 725, 726, 735, 736 3-(2'-fluoroethyl)spiperone labeling 478 8-fluoroganciclovir labeling 474 fluorohalobenzene labeling 266–7 4-fluoroiodobenzene labeling 267 fluorometaraminol labeling 206–7 fluoromethyl bromide labeling 258 fluoromethyl iodide labeling 258 fluorometoprolol labeling 542 fluoromexestrol labeling 207–8 fluoromisonidazole labeling 535, 536, 636 16|J-fluoromoxestrol labeling 725, 726 fluoronorepinephrine labeling 206, 263 8-fluoropenciclovir labeled 474 4-fluorophenacyl bromide labeling 697 2-fluorophenylalanine labeling 204 fluoropiperonal labeling 263 fluoroproline labeling 213 fluoropropionic acid methylester labeling 261–2 N-fluoropropyl-1p-carbomethoxy3pX4-iodophenyl)nortropane labeling 209 fluoropropyl bromide labeling 261 fluoropropyl iodide labeling 261 fluorotropapride labeling 263, 264 fluorotyrosine labeling 263 5-fluorouracil labeling 211 2-fluoroveratrole labeling 252 FP-TZTP labeling 490, 493–4, 496 gaseous targets 46 half-life 195, 248, 309, 725 hydrogen fluoride labeled 257 hydrogen labeling 257 imaging gene therapy 212–13 lomefloxacin labeling 210–11 ot-melanocyte-stimulating hormone labeling 663–4 metaraminol labeling 206–7 methyl 3-fluoro-5-nitrobenzimidate labeling 697 methyl lithium labeling 254 7V-methyIbenperidol labeling 208–9 N-methylspiperone labeling 607–8, 609 L-o-methyltyrosine labeling 635 monoclonal antibody labeling 262, 696–8 NCQ 115 labeling 264 neurotensin labeling 667 nitro groups 199–200 norepinephrine labeling 206 nuclear reaction 1% for production 248-50

nucleophilic fluorinating agent labeling 255–7 nucleophilic fluorination 197–200 octreotide labeling 650–1 peptide labeling 213 perchloryl fluoride labeling 251–2 perfluoroalkyl group labeling 201 PET labeled precursor production 248-68 phenethylamine labeling 206–7 phenylalanine labeling 203, 204 PK11195 labeling 544 positron emission 44–5 positron energy 248 potassium/Kryptofix system 197, 198 production 76, 196–7 reactions 45 proline labeling 213 propionic acid labeling 262 radioisotope separation 48 radiopharmaceuticals 195–6, 201–3 receptor ligand labeling 724–8, 729 setoperone labeled 594 solvent addition 198 specific activity 725 spiperone labeling 517 spiroperidol labeling 259, 260 substitution onto biomolecules 42 N-succinimidyl 1 -[(4'-fluorobenzylamino)]suberate labeling 697 jV-succinimidyl 4-fluorobenzoate labeling 262, 697 jV-succinimidyl 4-(fluoromethyl)benzoate labeling 697–8 N-succinimidyl 8-(4-fluorobenzyl)amino substrate labeling 262 sulfonamide labeling 255 targetry 45–8, 196–7 tetraalkylammonium salts 197, 198 tnmethylammonium groups 199– 200 tropane labeling 209–10 trovafloxacin labeling 210, 211 tryptophan labeling 203 vasoactive intestinal peptide labeling 658 water removal 198 xenon difluoride labeling 252-3 yield 314, 315 see also fluoride ions, fluorine-18 labeling 9-[(3-fluoro-1 -hydroxy-2-propoxy)methyljguanine (FHPG) 475–6 2'-fluoro-2'-deoxy-1 -p-D-arabinofuranosyl-5-iodouracil (FIAU) 432, 471-2 iodine-124 labeled 472 2-fluoro-2-deoxy-D-glucose, fluorine-18 labeling 252 l-fluoro-2-pyridone, fluorine-18 labeling 254 2-fluoro-3-[2(S)-2-azetidinylmethoxyJpyridine, fluorine-18 labeled 212 9-[4-fluoro-3-(hydroxymethyI)butylJguanine (FHBG) 475–6

833

INDEX 11 P- ftuoro- 5a-dihydrotestosterone 727 fluoro-16a-ethyl-19-norprogesterone, fluorine-18 labeled 208 21 -fluoro-16a-ethyl-19-norprogesterone (FENP), fluorine-18 labeled 737 3-fluoro-ot-methyltyrosine, fluorine-18 labeled 203–4 fluoro furanyl norprogesterone ketal (FFNP-ketal) 737–8 6-fluoro-L-dopa, fluorine-18 labeled 294-5 fluoro-i_-m-tyrosine, fluorine-18 labeled 205 Af-fluoro-jV-alkylsulfonamide, fluorine18 labeling 255 fluoroaeetone, fluorine-18 labeling 758 p-fluoroacetophenone 267 8-fluoroacyclovir, fluorine-18 labeling 474 fluoroacylating agents, fluorine-18 labeled 261-2 fluoroaianine, fluorine-18 labeled 263 fluoroalkanes, fluorine-18 labeling 258, 259 fluoroalkyl halides, fluorine-18 labeling 259 fluoroalkylbenzylsulfonate esters, fluorine-18 labeled 266 11 p-fluoroandrogens 727, 728 2-fluoroaniline, fluorine-18 labeled 252 2-fluoroanisole, fluorine-18 labeled 252 fluoroaromatics, fluorine-18 labeled 201 fluoroaryl precursors, fluorine-18 labeled 262-8 fluoroarylketones, fluorine-18 labeled 267-8 fluorobenzaldehydes fluorine-18 labeled 263, 264 reductive iodination 265 fluorobenzene-diazonium chloride, fluorine-18 labeled 267 4-fhiorobenzoic acid, fluorine-18 labeled 262 fluorobenzyl bromide, fluorine-18 labeled 264 fluorobenzyl iodide, fluorine-18 labeled 264 fluorobenzylalcohols, fluorine-18 labeled 263–4 fluorobenzylhalides, fluorine-18 labeled 263, 264–6 fiuorobenzyltrozarnicol, fluorine-18 labeled 264 4-fluorobromobenzene, fluorine-18 labeled 267 4-cis-fluorocaptopril, fluorine-18 labeling 761 fluorocarazolol, fluorine-18 labeling 542, 757–8 fluorocarboxylic acid, fluorine-18 labeled 261 fluorocatechol, fluorine-18 labeled 263 fluoroCGP12388, fluorine-18 labeled 542

fluorochlorobenzene, fluorine-18 labeled 267 fluorodemercuration 204 fluorodeprenyl, fluorine-18 labeled 263 fluorodestannylation 204 electrophilic 294–5 6-fluoroDOPA 252 fluorine-18 labeled 204–5, 263, 558 fluorodopamine, fluorine-18 labeled 206, 540 fluoroerythronitroimidazole (FETNIM) 536 fluoroestradiol, fluorine-18 labeled 287, 726, 727 16a-fluoroestradiol, fluorine-18 labeled 207, 725, 726, 735, 736 3-(2'-fluoroethyl)spiperone, fluorine-18 labeled 478 8-fluoroganciclovir, fluorine-18 labeled 474 fluorohalobenzenes, fluorine-18 labeled 266–7 4-fluoroiodobenzene, fluorine-18 labeled 267 fluorometaraminol, fluorine-18 labeled 206–7 fluoromethyl bromide, fluorine-18 labeled 258 fluoromethyl iodide, fluorine-18 labeled 258 (R,R)-fluoromethyl-QNB 492 (R,S)-fluoromethyl-QNB 492 fluoromethylbenzyl-4-phenylpiperazine 266 fluoromethylbenzyl spiperone 266 fluorometoprolol, fluorine-18 labeled 542 fluoromexestrol, fluorine-18 labeled 207–8 fluoromisonidazole (FMISO), fluorine18 labeled 515, 536, 636 16p-fluoromoxestrol (PFMOX) 735 fluorine-18 labeled 725, 726 fluoronorepinephrine, fluorine-18 labeled 206, 263 8-fluoropenciclovir, fluorine-18 labeled 474 4-fluorophenacyl bromide, fluorine-18 labeled 697 p-fluorophenacyl bromide 267-8 2-fluorophenylalanine, fluorine-18 labeled 204 fluoropiperonal, fluorine-18 labeled 263 fluoroproline, fluorine-18 labeled 213 fluoropropionic acid methylester, fluorine-18 labeled 261–2 jV-fluoropropyl-2p-carbomethoxy-3p(4-iodophenyl)nortropane, fluorine-18 labeled 209 fluoropropyl bromide, fluorine-18 labeled 261 fluoropropyl iodide, fluorine-18 labeled 261 3-(3-((3-fluoropropyl)thio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1 -

methylpyridine (FP-TZTP), fluor– ine-18 labeled 490, 493–4, 496 W-fluoropyridinium triflate 253 fluoroquinolones 210–11 fluorothia fatty acids, fluorine-18 labeled 533 fluorotrimethylstlane 256 fluorotropapride, fluorine-18 labeled 263, 264 fluorotyrosine, fluorine-18 labeled 263 5-fluorouracil, fluorine-18 labeled 211 2-fluoroveratrole, fluorine-18 labeled 252 fluoxetine 617 fluvoxamine 594 formaldehyde, carbon-11 labeled 1478, 153, 168 preparation 241–2 formoterol, carbon-11 labeled 542, 759 fractionation effects 771 free radical scavengers 123, 770, 785 frontal cortex, dopamine secreting neurons 585 o-fructose, carbon-11 labeled 153 fuzzy logic strategies 291, 294 G-protein-coupled receptor 345, 645 ligands 655 GABA acetylcholine modulation 618 analogues 161 dopamine interaction 610–11 dopamine modulation schizophrenia 612–15 substance abuse 611-12 extra-pyramidal motor system interactions 607 neurons 608 nitrogen-13 labeled 125 see also vigabatrin GABAA receptors 618 GABAergic interneurons 618 GABAergic ligands 161 galactose 803 P-galactosidase 469, 470 galactosyl-neoglycoalbumin (NGA), technetium-99m labeled 332, 333, 804–5, 806–7 receiver operating characteristic (ROC) 806-7 gallium 24, 25, 363-73 biodistribution 368 chelating agents 366, 367 chemistry 365-8 formation constants 365, 366 hard acids 367 hydrolysis 367 solubility 365, 366 tumor receptor imaging agents 734– 5 gallium-66 33–4, 364 physical characteristics 368–9 gallium-67 33, 34–5 biodistribution 371-2 carrier-free 363 cost 368-9

834 galHum-67 (contd) cyclotron production 368 decay 369 half-life 363, 364, 368 localization mechanism 370-1 normal distribution 370 octreotide labeling 650 photon efficiency 369 physical characteristics 368-9 preparation 369 radiation dosimetry 370 response to therapeutic intervention 370 scintigraphy 371 transferrin binding affinity 365, 367 uptake in tumor cells 370–1 uses 392 gallium-67 citrate 369-72 scintigraphy indications 370 gallium-68 35, 363, 364, 369 generator 369 localization 373 macroaggregate labeling 755 microaggregate labeling 755 octreotide labeling 650 PET use 392 physical characteristics 368–9 preparation 373 radiation dosimetry 373 scintigraphy 373 gallium-72 363 gallium oxide 24 gamma camera, dual detector 812-13 gamma rays emission 89, 92 prompt 89 gamma scintigraphy indium-Ill 702 radiopharmaceuticals 698 ganciclovir 473 8-ganciclovir, carbon-14 labeled 474 gas flow rate 292 gas targets 52, 78 gases for inhalation 52 gastrin 667-9 indium-Ill DTPA labeling 668 gastrin-I 668 gastrin-releasing peptide (GRP) 659, 660 gastrointestinal cancer, rhenium-labeled MAb 703–4 Gatterman reaction 431 GB67, carbon-11 labeled 161, 543 GBR-12909 607–8, 616 gene expression, imaging methods 469 gene knockout technology 739 gene therapy 467-79 imaging 212–13 surrogate markers 468 generalized linear least squares method 509, 511 modification 518-19 genetically modified animals 559 genome damage 767 germanium 25 half-life 392

INDEX germanium-68 24–5 germanium oxide 25 Gilles de la Tourette syndrome see Tourette syndrome glioblastoma, substance P receptors 659 globus pa Hid us 586 glomerular capillary 807–8 glomerular filtration 808 rate 809, 812-13, 814 glomerulus 810 glucagon, nitrogen-13 labeled 127 glucocorticoid receptors 738 glucoheptonate complex 333, 334 glucose carbon-11 labeled 167, 531 hepatic storage 797 metabolism in brain 307, 310, 311 cocaine abuse 567-8 phosphorylation 629, 630 tumor cell metabolism 630 D-glucose, carbon-11 labeled 153 glucose-6-phosphate 629, 630 glucose membrane transporters (GLUT) 630 glucuronidation pathway 798 GLUT 1 630 GLUT 3 630 L-glutamate 151 glutamate, nitrogen-13 labeled 123, 124 glutamate dehydrogenase 123 glutamate-oxaloacetate transaminase 124 glutamate-pyruvate transaminase 124 glutamine synthetase 124 glutathione 325 glycogen 797 glycogenolysis pathway 797 gold-198 colloid 799, 800 gold backing plates 28 gold foil 37 GP Ilb/lIIa receptor antagonists, technetium-99m labeling 339–41 granatane 346 granisetron, carbon-11 labeled 161 granulation cell FDG uptake 630 granulomas, somatostatin receptors 645 Grignard reactions 147, 241 carboxylic acid salts 148 Grignard reagents 146, 147, 241 reaction with carbon-11 labeled oxygen 243 growth factors 213 tumor survival/proliferation 669 GTP protein 589 guanidine, carbon-11 labeled 145 H-acidic compound radiofluorination 260 H-Tyr-D-Met(O)-Phe-Gly-NH2 127 habenula, dopamine secreting neurons 585 half-life determination 87 hallucinogen receptors, brominelabeled 453

halobenzenes, recoil labeling 266 halodestannylation reactions, electrophilic 724 halofluorination reaction 727, 728 halogens chemical properties 443 exchange labeling 429 halonitriles 149 carbon-11 labeled 148, 238 haloperidol 345 receptor binding 583 halophenyl-lrimethylammonium salts 267 HAMA 385 harmaline 163 harmine 163 head and neck cancer recurrence diagnosis 634 staging with FDG-PET 633 heart cocaine uptake 562, 563-4 fatty acid metabolism tracers 531–5 FDG uptake 629 glucose metabolism tracers 531 metabolic imaging 529 myocardial hypoxia imaging 535–7 myocardial perfusion tracers 529–31 myocardial receptor imaging 540–5 myocardial sympathetic nerve imaging 537–40 see also cardiac entries; myocardial entries heart agents 529–45 technetium 327–30 heart disease, 18FDG use 313 heat deposition 5 transfer 6 Heck reaction 155, 156 helium-3 36, 48 hepatectomy 806 hepatic angiograms 801 see also liver hepatic arterial flow 798 hepatic binding protein 803 hepatic blood flow measurement 799 hepatic metastases, indium-111OctreoScan 387 hepatic perfusion 799–801 hepatobiliary agents 331, 332–3, 801–3 hepatobiliary imaging, technetium99m-HIDA 325 hepatocellular functional mass 803-7 hepatocytes 797, 798 lipid metabolism 797 hepatoma, gallium-67 in detection/ staging 370 herpes simplex virus thymidine kinase (HSVl-tk) 213, 432, 470, 471–6 acylguanosine analogs 471, 476–7 FHBG production 475–6 FHPG production 475–6 ganciclovir 473, 474 gene expression imaging agents 476–7 intratumor injection 468

835

INDEX penciclovir 474 substrates 471–6 uracil analogs 471, 472, 476–7 heterocyclic compounds 148 hexamethylpropyleneamine oxime 325 hexestrol, iodine substitution 722 hexokinase 583 FDG phosphorylation 629 reaction 309 regional activity in brain 584 substrate specificity 309, 310 a-HIBA 108 high-pressure liquid chromatography (HPLC) 295 automated plasma assays 296–7 bromine 448 hepatobiliary agents 332 hippocampus, dopamine secreting neurons 585 histamine H-l receptors 163 histamine receptors, cimetidine binding 58? HL-91 331 technetium-99m labeled 537 Hodgkin's disease 337 gallium-67 in detection/staging 370, 392 holmium-l 16 reactor production 107-8 specific activity 108 hormones, peptide 213 hot atom chemistry 42–3, 231 excitation labeling 428 5-HT receptors activity 609 cortical 609 /V-methyl spiperone binding 506 vapreotide affinity 653 5-HTj receptors 589 5-HT,A receptors 160, 590 depression 593–4 technetium-99m labeled 343–4 5-HT2 receptors 161, 589 jV-methylbenperidol selectivity 208 selective antagonists 212 5-HT2A receptors 591 Alzheimer's disease 593 clozapine occupancy 594 depression 594 schizophrenia 591, 594 technetium-99m labeled 343–4 5-HT3 receptors 161, 590 5-HTTLPR short allele 592 HuM195 monoclonal antibody 704 human chorionic gonadotrophin (hCG), p-chain 468 Huntington's disease 610 hydraulic tube facilities 102, 106–7 hydrazino nicotinamide 336 hydrobromic acid 264 hydrochloric acid 316, 317 hydrogen fluorine-18 labeling 257 free radical scavenger 123 hydrogen-3 cocaine labeling 560

compounds 489 decay 778 displacement of compounds by nonradioactive derivative 491-2 raclopride labeling 506 WIN 35428 labeling 560 hydrogen cyanide, carbon-11 labeled 145, 148, 149 hydrogen fluoride carrier 257 fluorine-18 labeling 257 hydrogen peroxide 430 oxygen-15 labeled 131–2 hydroiodic acid 264 5-hydroxy-L-tryptophan (5-HTP) 151 carbon-11 labeled 164 TV-hydroxysuccinimide 125 hypobromous acid 447 hypoiodite ions 424 hypothalamus imaging 738 hypoxia, cellular 770 hypoxia agents copper-labeled 410–12 technetium labeling 330–1 ICI 118,551 759 imaging gene therapy 212–13 imaging methods, high resolution 739 imidazoles derivatives 636 fluorinated 536 imidazolidinone 166 4-imidazolyl group, carbon-11 labeled 159 iminodiacetic acids, technetium-99m labeled 802–3 2-iminothiolane (2IT) 413 immunoglobulin G (IgG) 686–7 constant (C) domains 687 heavy (H) chains 687 labeling 688 light (L) chains 687 variable (V) domains 687 immunoglobulin Ga (IgGj), hinge region 688 immunoglobulin reagents 427 incident particle 89, 90, 92 charged 91 kinetic energy 92 indium 363, 364–5, 373–92 chelating agents 376 chemistry 373–6, 377 formation constants 373 hydrolysis 374 physical characteristics 377-8 solubility 374 stability constants 374 transferrin binding 374-5 indium (IV) 375 indium-110 39-40 indium-Ill 40,364–5 Auger electrons 378, 389, 647 cost 377 cyclotron production 377 DOTA cholecystokinin labeling 669 DOTA-lanreotide labeling 654

DTPA 364, 375, 390–1 DTPA-B72.3 labeling 702 DTPA-octreotide 337 gastrin labeling 668 8-hydroxyquinoline [oxine] 364 imaging characteristics 378 monoclonal antibody labeling 701, 702-3 MyoScint labeling 365, 390 nuclear characteristics 374 OctreoScan labeling 365, 386–9 octreotide labeling 337, 647–9 OncoScint labeling 364–5, 385–6, 412 oxine 378, 379, 382 photons disintegration 369 yield 378 platelet labeling 382 ProstaScint labeling 365, 377, 382-4, 412 targeted radiotherapy 644 transferrin binding affinity 365, 376 tumor detection 392 white blood cell labeling 377, 378–80 Zevalin labeling 393 indium-lll-WBC 377, 378–80, 392 dosimetry 379, 380 half-life 380 localization 380 preparation 378–9 scintigraphy 380 indium-113m 377 nuclear characteristics 374 transferrin 762 indium-114m 377 cyclotron produced 378 lymphocytes 391-2 nuclear characteristics 374 oxine 391 proton energy 378 targeted radiotherapy 644 indium3+ 375–6 indocyanine green labeling 428 infection imaging agents 339 inflammation, airway neural response 751 inflammation imaging agents 339 gallium-67 citrate 370 inflammatory cells, FDG uptake 630 inflammatory response, otvPs integrin 671 influenza virus neuraminidase inhibitor 163 insulin iodine-123 Tyr labeled 804 nitrogen-13 labeled 127 receptors 582 a v p 3 integrin 670-3 peptide ligands 671–3 integrin receptors 338 integrins 670–4 intracellular process activation 670 RGD sequence 670, 671–3 tumor uptake 671, 672, 673 P.i integrins 671

836 inter-halogen compounds 424 internal conversion 775 intranuclear cascade 91 iodide carrier-added 427 labeled 428 iodide-iodate system 430 iodinated compounds structural integrity 433 uses 434 iodination/iodination reactions chemical oxidizing reagents 429–31 regio-specific 427 ring 430 synthetic techniques 434 iodine 423-4 bromine comparison 443–4 carrier-free species 427 cationic species 424 elemental 429, 430 equilibrium constant 424 excitation labeling 428 halogen exchange labeling 429 hypophalous acid 423–4 ions 428 isotopic exchange 429 nuclear medicine applications 424–5 organoboranes 432 organometallic precursors 432 organostannanes 432–3 radioisotopes 425–7, 721–4 half-lives 423 radiolabeling 427–8 regio-specific reactions 431–2 solubility 423 iodine-120g 17 iodine-121 17–18 iodine-122 425 iodine-123 4, 18–22, 425, 434 p-CIT labeling 592-3, 594 clonal survival 784 effectiveness 777 estrogen labeling 735 5-iodo-6-nitro-2-piperazinylquinoline labeling 591 15-/>-iodophenyl-3-(R,S)-methylpentadecanoic acid labeling 532-3 monoclonal antibody labeling 690 orthoiodohippuran labeling 811 PK11195 labeling 544–5 production reactions 18-20 5-I-R91150 labeling 592 radioisotope separation 22 radiolabeling 428 somatostatin analog labeling 646 targetry 20–2 Tyr-indulin labeling 804 vasoactive intestinal peptide labeling 656 iodine-124 4, 22–3,425, 431–2, 434 FIAU labeling 472 2'-fluoro-2'-deoxy-1 -p-o-arabinofuranosyl-5-iodouracil labeling 472 monoclonal antibody labeling 690 positron emission 724 iodine-125 98–9, 434

INDEX 3-acetamido-5-iodoproflavine labeling 784 Auger electron emission 783–6 decay 785 displacement by non-radioactive derivative 491-2 DNA-incorporated 785, 786 effectiveness 777 iododeoxyuridine labeling 784, 785, 786 5-(2-iodovinyl)-2'-deoxyuridine labeling 472, 473 linear energy transfer 772 monoclonal antibody labeling 690 radiotoxicity 784 survival curve 779 iodine-125 metaiodobenzoate, bombesin labeling 660 iodine-130 88 iodine-131 88, 90, 100, 425, 434 albumin microaggregates 801 cost 689 decay 778 Diodrast 426 distribution in body 774 gastrin-I labeling 668 linear energy transfer 772 meta-iodobenzylguanidine labeling 777 monoclonal antibody labeling 68990, 779–80 orthoiodohippuran labeling 811 Rose Bengal labeling 332, 801-2 iodine monochloride 427, 431 carrier-free 428 iodine oxides 423 5-iodo-6-nitro-2-piperazinylquinoline, iodine-123 labeled 591 iodo species 424 iodoaryl derivatives 432 m-iodobenzylguanidine 162 iododemetallation reactions 432 iododeoxyuridine (IUdR) 777 iodine-125 labeled 784, 785 experimental radionuclide therapy 786 iododestannylation 433 16ct-iodoestradiol 735 iodoestradiols, radiolabeled 429 lodogen 427, 430, 431 0,m./Modohippuric acid 429 iodomethane, carbon-11 labeled 297 15-/7-iodophenyl-3-(R,S)-methyIpentadecanoic acid (BMIPP), iodine123 labeled 532-3 />-iodophenylalanine 429 3-iodopropyl-1-triflate 261 iodopyridine 692 iodostannylation 432 iodothienyl derivatives 432 iodouracil 785 iodovinyl 692 5-(2-iodovinyl)-2'-deoxyuridine (IVDU), iodine-25 labeled 472, 473 iodovinyl derivatives 432

17ot-iodovinyl estrogens 722 IQNP-125 454 iron binding proteins 367 biodistribution 368 blood-to-brain transport 29 physiologic stable states 368 transferrin saturation 371-2 uptake 371 iron-52 29 iron-54 30 iron-55 30 irradiation carbon traces 44 PET isotopes 42 power deposition 5–6 target 42 see also radiation ischemia, hypoxia imaging 330 isocarbacyclin methyl ester 154, 155 isomerase enzyme inhibition 796 isoprenaline 759 isotopes see nuclides isropidine, carbon-11 labeled 163 K-receptor ligands 162 ketamine 613 carbon-11 labeled 163 ketanserin 344, 609 ketones 149 carbon-11 labeled 156 kidneys cocaine uptake 562, 563 collateral circulation 808 function 807-10 see also renal entries kinetic energy 2, 42 transfer to nucleus 88, 89 Kontron industrial microcomputer 285 Kryptofix 2.2.2. 238, 253, 257, 258, 262, 263 18 FDG synthesis 287, 314–15, 316, 317 fluoride ion fluorine-18 labeling 31415, 316 krypton, Auger electrons 446 krypton-75 444 krypton-76 445 electron capture decay 446 krypton-77 16, 445 P* decay 446 electron capture decay 446 krypton-Sim 752, 753 Kupffer cells 800, 801 radiopharmaceutical extraction 798 reticuloendothelial system 797 L-703, 717 162 lac Z reporter gene 469 lactoperoxidase 427. 430 lanreotide 654 lanthanide contraction 108 lanthanum 36 lead-208 704 Lesch-Nyhan disease 589

837

INDEX leukemia lymphocytic 372 therapy 777 leukokinin 339 LeuTech 392 levophanol 583 Licostinel 158 ligand-protein interface 719 ligands affinity 718 competitive 604–5 deformable pockets 719 irreversibly binding 508, 515–16, 520, 584 modeling options 513–14 non-radioactive 489, 490–1 labeled compound displacement 491–2 noncompetitive 606–7 PET imaging 604-5 preformed pockets 719 reversible 508-12, 584 measured plasma input function 508-10 small organic 716 transport from plasma to tissue 513 see also radioligands limbic cortex, dopamine secreting neurons 585 linear energy transfer 771-2 alpha particle emitters 781 astatine-211 695 high 767, 772 low 767, 768, 772 linear least squares analysis 509, 511 liphophilic drug catabolism 798 lipid metabolism 797 liquid detectors 292 liquid targets 78 lithium alky 1 compounds 146, 147 lithium aluminium hydride 125, 146, 239, 241 lithium dialkyamides 155 lithium methyl(2-thienyl)cuprates, carbon-11 labeled 157 liver blood flow 797 cocaine uptake 562, 563 functional mass index 806 image interpretation 800–1 intestinal substance uptake/ redistribution 797-8 phagocytosis 797 plasma flow 797 sinusoid blood flow/volume periodicity 797 target receptor for imaging 803–4 see also hepatic entries liver disease 331 end-stage 800, 801 liver function diagnostic parameters 806 evaluation 798-807 measurement 797–8 radiopharmaceuticals for study 795– 807

reserve prior to hepatectomy 806 transjugular intrahepatic portosystemic shunt (TIPS) 805–6 liver tumors, computed tomography (CT) 801 L,L-TcO-ECD 326, 327 Logan Plot 559 logic controllers, programmable 296 lomefloxacin, fluorine-18 labeled 210–11 loop method for PET radiotracers 297 loop of Henle 809–10 lorazepam 610–11 fluorine-18 labeling 259, 260 lung(s) P-adrenoceptors 755–9 amine metabolism/pharmacokinetics 762 endothelial damage 762 muscarinic receptors 760–1 pulmonary function imaging 751–62 serotonin 762 see also pulmonary entries lung agents 331-2 lung cancer copper-62-ATSM 412 disseminated 635 FBAU as proliferation marker 451–2 gallium-67 in detection/staging 370 lung cancer, non-small-cell 337 diagnostic FDG-PET 631, 652 FDG uptake 629, 630 staging 632-3 substance P receptors 659 lung function 751 PET 752 lung perfusion 751 gases 752–4 lutetium-176 109 lutetium-177g 108-9 Lym-1 monoclonal antibody 413 lymph node agents 331 lymph nodes, sentinel node imaging 331 lymphocytes indium 114m-labeled 391–2 somatostatin receptors 645 lymphoid cancers 715 lymphoid tissues, glucocorticoid receptors 738 lymphoma "TDG in diagnosis/staging 392 gallium-67 in detection 392 glucocorticoid receptors 738 indiurn-111 DTPA octreotide 648–9 recurrence diagnosis 634 therapy 777 see also Hodgkin's disease; nonHodgkin's lymphoma lymphoscintigraphy 331 lysine, monoclonal antibody V domain 688-9 [Iysine3]bombesin 661 lysosomes 648 macromolecules bromine-76 labeled 456

iodination 430-1 magnesium-22 27 magnesium-23 27 maleimido bond 694, 695 manganese 29 manganese-51 28 manganese-52m 29 manganese oxides 446 mass excess 91 maximum tolerated dose (MTD) 771 McN5652, carbon-11 labeled 591.592 MDMA 592 MDR1 gene 347 mebrofenin, technetium-99rn labeled 803 ot-melanocyte-stimulating hormone (MSH) 662-4 analogs 663–4 fluorine-18 labeled 663–4 melanoma cell binding 662, 664 radiolabeling 663 ot-melanocyte-stimulating hormone (aMSH) receptor 338, 662, 663 melanoma bromine-76-UdR 451 18 FDG in diagnosis/staging 392, 633 galliurn-67 in detection/staging 370, 392 Ps integrins 671 ot-melanocyte-stimulating hormone binding 662, 664 a-melanocyte-stimulating hormone receptors 662 melatonin, carbon-11 labeled 166 membrane receptor structure 719-20 mercaptoacetyldiglycyl-y-aminobutyric acid (MAG2-GABA) 704 mercaptoacetyltriglycine (MAG3), technetium-99m labeled 704, 809, 811 mesolimbic pathway, dopamine secreting neurons 585 mesterolone, carbon-11 labeled 157, 167 meta-iodobenzylguanidine (MIBG) 537-8 analogs 538 iodine-131 labeled 777 metabolic imaging, heart 529 metabolic radiotherapy 426 metahydroxyephidrine, carbon-11 labeled 538–40 metal complex design 698 metals 29-42 metaraminol carbon-11 labeled 161, 538, 519 fluorine-18 labeled 206–7 metastases hepatic 387 skip 384 staging with FDG-PET 633 methane 48, 49, 50 carbon-1 ilabeled 145, 146, 148 over pressure 123 methane sulfonates 147

838 methane sulfonyl chloride, carbon-11 labeled 147 methanethiol, carbon-11 labeled 246 methanol, carbon-11 labeled 148 methionine, carbon-11 labeled 153, 165, 245, 634 methotrexate co-administration with 125 IUdR 786 11 p-methoxy-17a-iodovinylestradiol 735 methoxyfluoride 730 1 -(2-methoxyphenyl)piperazine (MPP) 343, 344 methoxyprogabic acid 125 5-methyl-2'-fluoroarabinouridine (FMAU) 471 methyl 3-fluoro-5-nitrobenzimidate, fluorine-18 labeled 697 jV-methyl-4-piperidyl benzilate 162 methyl bromide, carbon-11 labeled 147 N-methyl-D-aspartate (NMDA) receptor complex ligands, carbon-11 labeled 162-3 methyl iodide carbon-11 labeled 147–9, 154, 155, 165, 168, 243 loop method 297 preparation 239–40 tumor receptor ligands 731 recoil labeling 240 methyl lithium carbon-11 labeled 147, 240, 247, 730 fluorine-18 labeled 254 methyl magnesium iodide 241 methyl p-hydroxybenzimidate hydrochloride 427 N-methyl-piperidin-4-yl-2-cyclohexyl2-hydroxy-2-phenylacetate ((R)VC-002), carbon-11 labeled 760–1 methyl triflate, carbon-11 labeled 1467, 240–1

loop method 297 methyl trifluoromethanesulfonate see methyl triflate methylaminobenovesamicol 162 methylating agents, carbon-11 labeled 239–1 N-methylbenperidol, fluorine-18 labeled 208–9 methylene iodide 22 carbon-11 labeled 145, 146–7 l-(3-methylheptadecanoic acid, carbon11 labeled 532 methylhypofluorite, carbon-11 labeled 147 methylisocyanate, carbon-11 labeled 148 methylmagnesium iodide 147 methylnorapomorphine 160 methylphenidate abuse 565 carbon-11 labeled 517, 519, 559, 565 clearance rate 565 cocaine comparison 564-5 dopamine receptor levels 572

INDEX enantiomers 564 pharmacokinetics 565 time-activity curve in brain 566 W-methylpiperidine-4-yl acetate 163 methylsergide 617 Af-methylspiperone 506 carbon-11 labeled 297, 584, 587, 591, 594 fluorine-18 labeled 607-8, 609 W-methylspiroperidol carbon-11 labeled 514, 605 radiolabeling 230 methyltetrahydroaminoacridine 163 L-a-methyltyrosine, fluorine-18 labeled 635 metoprolol, carbon-11 labeling 541 microcomputers 286–7 microdialysis 559 microwaves 142 milameline 162 mineralocorticoid receptors 719 miniaturization 142 minigastrin, indium-Ill DTP A 668 mitochondrial complex I activity 164 mitochondrial electron transport enzyme 410 MK-801 506 moclobemide 571 moderator 93 molecular weight 491 molybdenum-94 37, 38 molybdenum-99 104–6 fission produced 104–5, 706 neutron capture 105–6 non-fission production 105-6 production disadvantages 105 specific activity 105–6 technetium-99m generation 698 molybdenum-99/technetium-99m generator system 88, 90, 100, 104–6 molybdenum oxide 38 mono-oxotechnetium(V) complex 326 monoamine oxidase 539–40 inhibition 569–71 platelet levels in smokers 569, 571 tobacco smoke exposure 568-71 monoamine oxidase inhibitors (MAOIs) 505 monoamine oxidase type A enzyme 163 monoamine oxidase type B enzyme 163 monoamines, reuptake sites 586 monoclonal antibodies 685 accumulation in tumors 686 amino acid reactivity 689 antigenic target 685, 686 astatine-211 labeled 695–6 bismuth labeling 704 bromine labeling 456–7 chimeric 688 copper labeling 404, 412–14, 699 dehalogenation 689 deiodination 691 fluorine-18 labeling 262, 696–8 hinge region flexibility 688 humanized 688

indium-Ill labeling 702-3 internalizing with radiometals 686 iodine-131 labeling 689–90 iodine radionuclide labeling 426, 690 labeling 43–4, 643, 690 metal radionuclide labeling 698–704 bifunctional chelating agents 698–9 murine origin 687–8 radioiodinated 689 direct electrophilic methods 691 internalizing 692–4, 695 oligosaccharide conjugates 693 positively charged templates 6934 yV-succinimidyl iodobenzoates 691-2 rhenium labeling 703–4 stability 688 technetium-99m labeling 701-2 therapy 689 yttrium-90 labeling 689–90. 700–1 MPP* receptor binding 586 p-MPPl 343 MQNB, carbon-11 labeled 544 mRNA imaging probe 469, 478 MRP-12 complex 326, 327 MRPl gene 347 MRP1 protein 348 multidrug resistance cancer cells 335, 347-9 reversal agents 348, 349 multidrug-resistance-associated protein (MRPI) 347 muscarinic acetylcholine receptors 162, 487 corpus striatum 607 M2 receptors 492, 493–4 subtype selectivity determination 491-2 muscarinic agonists, selective M2 490 muscarinic cholinergic receptors, bromine labeling 454 muscarinic receptor-binding radiotracers 487–96 muscarinic receptors 760–1 myocardial 543–4 schizophrenia 615 subtypes 760 myeloperoxidase 448 myocardial hypoxia 529 tracers 535–7 see also heart myocardial infarction hypoxia imaging 330 MyoScint 365 myocardial metabolism, brominelabeled fatty acids 449 myocardial perfusion copper-62-PTSM 408–9 imaging 327, 328. 330, 407 tracers 529–31 myocardial receptor imaging 540–5 myocardial sympathetic nerve imaging 537–40 MyoScint 702 i n d i u m - I l l labeled 365. 390

839

INDEX [13N]ammonia 120 NCQ 115, fluorine-18 labeled 264 Nefs reaction 244 neon-19 753 neon-20 45, 46 neon-20{d,a)fluorine-18 reaction 558 neon gas, deuteron irradiation 197 nephron 807, 809, 810 nerve terminals, pre-synaptic 586 neural crest tumors, iodinated MIBG 777 neuroendocrine tumors indium-Ill DTP A octreotide 648-9 OctreoScan 365, 387 yttrium-90 labeled peptides 777 neurofibrillary tangles 488 neurokinin-1 receptors 163 neurokinin A 751 neurons dopaminergic 585, 608 post-synaptic 586 GABA 608 post-synaptic dopaminergic 586 neuroreceptor binding 585 striatal 616 substantia nigra dopamine secretion 585 neuropeptide Y (NPY), nitrogen-13 labeled 127 neuropeptides 645 lung function 751 radioligands 755 neuropsychiatric disorders, dopamine receptors 584 neuroreceptor agents 341–4, 345–7 neuroreceptor ligands carbon-SI labeled 729 technetium-labeled 732 neuroreceptors 582–3 dynamic properties 603 transport system 732 see also dopaminergic system; serotonergic system neurotensin receptors 666 neurotensins 666–7 analogs 667 fluorine-18 labeled 667 neurotransmitters competition 505 drug-induced changes 505 dynamic interactions 603–19 endogenous concentration 505 kinetic modeling 605–6 PET measurement of interactions 606–7 neutron capture 71, 96, 97 cross-section 94, 95, 96 decay of intermediate radionuclide 98–9 metastable nuclei production 106, 107 molybdenum-99 105–6 neutrons bombardment 89 epithermal 89

fast 89, 99 flux 93, 94 inelastic scattering 106, 107 production approaches 96–100 resonance 89 thermal 89, 92, 93 fission 90 flux 95 nickel 29, 46 alloys 24, 46 target for copper radionuclide production 403 nickel-60 32 nickel-61 31 nickel-gallium alloys 24 nicotine 569, 571 brain changes 568 dopamine Dj receptor occupancy 612 nicotinic acetylcholine receptor, bromine labeled 453–4 nicotinic cholinergic receptors 162 nicotinic receptors, radiotracers 212 nigro-striatal pathway 583 dopamine secreting neurons 585 niobium-90 targeted radiotherapy 644 nisoldipine, carbon-11 labeled 163 nitrate, nitrogen-13 labeled 121 nitrate ions 52 nitric oxide 751 nitrogen-13 labeled 126 nitric oxide synthase 164 nitrido dithiocarbamate complex 530 nitriles 149, 152 nitrite, nitrogen-13 labeled 121-2. 127 nitrite ions 52 P-nitroacetophenone 267 nitroalkanes 147 carbon-11 labeled 244–5 nitroethane, carbon-11 labeled 245 nitrogen atmospheric 87 natural 53 nitrogen-13 labeled 121, 754 oxo anions 122–3 nitrogen-13 42 amine labeling 125–6 amino acid labeling 123–5 aminodecane labeling 125, 726 aminohexane labeling 125 aminooctane labeling 125 antiturnor drug labeling 126–7 asparagine labeling 125 carbon dioxide labeled 754 chemistry 119, 120–8 cisplatin labeling 127 dopamine labeling 125 glucagon labeling 127 glutamate labeling 123, 124 lung function 753, 754 neuropeptide Y labeling 127 nitrate labeling 121 nitrite labeling 121–2, 127 nitrogen dioxide labeling 126 nitrosamine labeling 127 nitrosocarbaryl labeling 127

nitrosothiol labeling 127 jV-nitrosourea labeling 126 nitrous oxide labeling 122 phenethylamine labeling 125 phenylpropionamide labeling 125 positron emission 51–2 production 76, 120 reactions 51 putrescine labeling 125 radioisotope separation 52 radiopharmaceuticals 119 radiotracer 44 secretin labeling 127 streptozotocin labeling 127 substitution onto biornolecules 42 targetry 51-2 urea labeling 126 vasoactive intestinal peptide labeling 127

see also ammonia, nitrogen-13 labeled nitrogen-14(p,a)carbon-11 reaction 558 nitrogen-15 53 nitrogen/carbon dioxide mixtures 12930 nitrogen/carbon monoxide mixtures 130 nitrogen dioxide nitrogen-13 labeled 126 oxygen-15 labeled 130 nitrogen gas 49, 50 carbon-11 production 44 nitrogen/hydrogen mixtures 130 nitrogen/oxygen mixture irradiation 128-9 nitrogen/water vapor mixtures 130 2-(2-nitroimidazol-1 [H]-yI)-N-{3fluoropropyl)acetarnide (EF1) 535, 536 nitroimidazole compounds 410, 535, 536–7 nitromethane, carbon-11 labeled 244, 245 |3-nitrophenethyl alcohol 245 nitropropane, carbon-11 labeled 245 nitrosamines, nitrogen-13 labeled 127 Ar-[13N]nitroso-N-chloroethyl-l-chloroethyl carbamate see BCNC nitrosocarbaryl, nitrogen-13 labeled 127 nitrosothiols, nitrogen-13 labeled 127

nitrosoureas, carbon-11 labeled 168 N-nitrosoureas, nitrogen-13 labeled 126 nitrostyrene, carbon-11 labeled 244 nitrous oxide 430 nitrogen-13 labeled 122 NK1 658 NK2 658 NK3 658 NMSP, carbon-11 labeled 514 NNC 13-8199, bromine-76 labeled 454-5 NNC 22-00100, bromine-76 labeled 456

840 NNC 112, carbon-11 labeled 517 nofetumomab merpentan, technetium99 labeled 701 nomifensine 160 non-Hodgkin's lymphoma 337 copper-67 labeled monoclonal antibodies 413 gallium-67 in detection/staging 370 staging with FDG-PET 633 nonlinear least squares approach (NLLSQ) 502, 517, 520 norepinephrine analogs 537 carbon-11 labeled 167, 245 fluorine-18 labeled 206 reuptake 586 transporter 163 cocaine binding 560, 564 transmembrane domains 586 norepinephrine receptors, bromine labeled 453 norepinephrinergic neurons, Parkinson's disease 592 norphenylephrine, carbon-11 labeled 245 NR-LU-10 monoclonal antibody 7001, 702 nuclear elastic scattering 88 nuclear excitation energy 89 nuclear fission 90 reactors 93-5, 96 spontaneous 90 nuclear force, short-range 88 nuclear hormone receptors imaging 737–8 sequence comparisons 719 structure 718–19 nuclear inelastic scattering 89 Nuclear Interface machine 295 nuclear particles, sources 93–5, 96 nuclear reactions definition 89 endoergic 2, 91 energies used 1, 3, 91, 94 exoergic 2, 91 fission products 94 knock-on 90–1 minimum energy 3 models 1–2, 89–91 notation 89 number of reactions occurring in one second 3 probability 91–2 production equation 92-3 projectile-target processes 88–9 saturation 92-3 nuclear reactors control rods 94 core 94 energy spectrum 94 fuel 94 fuel rods 95 power production 94 research 93, 95, 96, 97 nuclear resonance 92 nuclear vacancy 775

INDEX nucleic acid chemistry, automated 284–5 nucleons 90 nucleosides, ring-closure reactions 158 nucleus de-excitation 90, 92 decay channels 2 excitation energy 2, 92 number per unit area 3–4 radius 91 target 92 nucleus accumbens dopamine reuptake inhibitors 611 dopamine transporter protein distribution 586 nuclides 87 see also radionuclides octopamine, carbon-11 labeled 167 OctreoScan, indium-111 labeled 365, 386–9 dosimetry 387 localization 387-9 preparation 386 scintigraphy 386–7 octreotate, tyrosine3 651–2 octreotide 386, 414, 645–6 copper-64 labeling 651 DOTA labeled 389 fluorine-18 labeling 650–1 gallium labeling 650 glycated analogs 652-3 indium-Ill DTPA 647–9 clinical use 648-9 internalizing in tumor cells 647 metabolites 648 renal metabolism 647–8 somatostatin receptors 648 tumor imaging 657 indium-Ill labeled 337 labeling for PET 650–1 radiolabeled 644 technetium-99m labeled 649 tyrosine-3 analog 646, 651 see also OctreoScan octreotide-14 645, 646 octreotide-28 645, 646 olanzapine 594 olfactory tubercle, dopamine transporter protein distribution 586 oligodeoxynucleotides 469, 478, 479 oligopeptide chemistry, automated 284–5 on-column preparations 142 on-line procedures 142 OncoScint, indium-111 labeled 364–5, 385–6, 412, 702 one-pot procedures 142 opioid receptor ligands, carbon-11 labeled 162 optical model 90 orhitofrontal cortex, cocaine abuse 568 organnostannanes 432–3 organoboranes amination 125, 126 radioiodination 432

organocuprates 154 ornithine, carbon-11 labeled 165 orthogonality 796, 806, 814 orthoiodohippuran, iodine-123 and iodme-131 labeled 811 orthophosphates 777 ovarian cancer ascites tumor 777 CA125 385 recurrence diagnosis 365, 634 steroid receptors 346 oxides boron-10 targets 234 carbon-11 labeled 233–5 nitrogen targets 234 oxine indium-Ill labeled 378, 379, 382 indium-114m labeled 391 oxygen ot-bombardment of flow 753 carbon-11 labeled 233–5,247 cellular effect 770 labeling 52 oxygen-15 labeled 53, 535 pulmonary transport 751 oxygen-15 42 bolus delivery 133 carbon dioxide labeling 131 carbon monoxide labeling 131 chemistry 119, 128–33 hydrogen peroxide labeling 131–2 nitrogen dioxide labeling 130 oxygen labeling 53, 535 ozone labeling 130 positron emission 52-3 production 76, 128–30 reactions 52-3 proton machines 80 quality control of labeled compounds 132 radioisotope separation 53 radiopharmaceuticals 119 radiotracer delivery 133 steady state intravenous infusion 133 substitution onto biomolecules 42 tandem cascade accelerator 79 targetry 53 waste management 133 water labeling 53, 130, 131, 530–1, 811 oxygen-16 51 oxygen- 16(ot,n)neon-19 reaction 753 oxygen-16(p,o)nitrogen-13 reaction 754 oxygen-18 carbon dioxide enriched 255 enriched target 47, 196, 197 proton reaction 45 water labeling 47, 196, 197, 250, 255, 314 oxygen-!8{p,n)fluorine-18 reaction 314, 315, 558 ozone 53 carbon-11 labeled 243 oxygen-15 labeled 130 ultraviolet photochemical reaction 242

INDEX P-glycoprotein 335 P280 341 P450 reductase 636 P748 341 P829 337 P14IO 665–6 PI666 337, 658 P4835H 339 PAC1.1 monoclonal antibody, technetium labeling 341 Paget's disease, calcitonin accumulation 664, 665 palladium 153–5 carbonylation 156–7 catalyst 237 cross-couplings 154–5 cyanations 154 Heck reaction 155 palladium-103 38–9 1-palmitate, carbon-11 labeled 532 (d-palmitate, carbon-11 labeled 5323 pancreatic cancer bombesin uptake 662 cholecystokinin uptake 669 depreotide uptake 655 neurotensin receptors 666 staging with FDG-PET 633 substance P receptors 659 pancreatic mass, diagnostic FDG-PET 631

parametric images 519–20 PARK-2 gene 587–8 parkin gene 587–8 parkinsonism, autosomal recessive juvenile (AR-JP) 587–8 Parkinson's disease 569, 587–9 depression 592 dopamine levels 582–3, 586 dopamine neuron loss 588 dopamine receptors 584 imaging 585 dopamine transporter protein (DAT) 588, 589 candidate gene 587 GABA action 610 L-DOPA 583 norepinephrinergic neurons 592 serotonergic system 592–3 SSRI treatment of depression 592 ubiquination abnormalities 588 particle acceleration principles 73–5 particle orbit 74 passivation 46 Patlak Plot 559 penciciovir 474 penicillamine 332 peptides brornine-76 labeled 456 carbon-11 labeled 166 copper labeled 404, 414–15 fluorine-18 labeled 213 iodine labeled 426 radioiodination 429–30 targeted radiotherapy 644 tumor imaging 643–75

841 perchloryl fluoride, fluorine-18 labeling 251-2 perfluoroalkyl groups, fluorine-18 labeled 201 peritubular capillaries 808, 810 permeability coefficient 491 peroxisome proliferator-activated receptor gamma (PPARy) 723 cancer growth regulation 738 peroxisome proliferator-activated receptor gamma (PPARy) receptor 727, 729 peroxisome proliferator-activated receptor ligands 715 pertechnate 323, 324 Pgp transmembrane protein 347 transport substrate 348 phagocytosis 797 phencyclidine 612-14 phenethylamines carbon-11 labeled 244 fluorine-18 labeled 206-7 nitrogen-13 labeled 125 phenolic compounds aryl radiofluorination 258 iodine reaction 430, 431 phenyl lithium 251 phenylalanine carbon-11 labeled 152, 165 fluorine-18 labeled 203, 204 phenylalanine dehydrogenase 124 phenylalkyl compounds, thallation 433 phenylephrine, carbon-11 labeled 162, 538–40 1 -phenylpiperazine 266 phenylpropionamide, nitrogen-13 labeled 125 phosgene 146 carbon-11 labeled 150, 161, 246–7 (S)-CGP 12177 production 756 preparation 242-3 phosphatidyl serine 336 phosphonates 333, 777 phosphonium salts, carbon-11 labeled 247-8 phosphorus 87 phosphorus-32 88, 773 chromate phosphate colloid 799, 800 phosphorus-33 773 photons 767 gamma 774, 775 oxygen effect 770 x-ray 775 phthalimide synthesis 156–7 physostigmine 163 phytate, technetium-99m labeled 799 pindolol 148, 756–7 pituitary adenylate cyclase activating polypeptide (PACAP) 655 PK11195 544–5 plasma automated analysis 296, 297 clearance rate 510 distribution volume 510, 517 measured input function 519 radioactivity 502

platelet factor 4 (PF-4) 339 platelets aggregation inhibition 341 disorders of destruction/production 381 indium-Ill labeled 382 monoamine oxidase 569, 571 serotonin uptake 593 platinum-195m antitumor drug labeling 106 reactor production 106–7 PLC, automatic synthesis of radiotracers 288 pneumatic tube facilities 101-2 Pneumocystis carinii pneumonia (PCP), gallium-67 citrate 370 polonium-211 695 polonium-212 704 polonium-219 696 poly (ADP-ribose) synthase 163–4 portal plasma flow 798 portal vein 797 positron emission tomography (PET) bromine-76 443-4 carbon-11 141–2 carbon-11 labeled methionine 634–5 cocaine studies 560–8 copper radiopharmaceuticals 401–2 cyclotrons 73 drug abuse research 557-73 dynamic with FDG 520 enzyme study compounds 159–64 FBAU as proliferation marker 451, 452 fluorine-18 44–5, 201–3 gallium-68 392 imaging data quantification 502-21 graphical analysis 509–10, 518–19 model selection 516-17 modeling options for irreversible ligands 513–14 modeling options for reversible ligands 508–12 outcome measure sensitivity 51516 parametric images 519-20 reference tissue methods 517–18 labeled precursor production 231-2 carbon-11 labeled 232–48 fluorine-18 labeling 248–68 lung function 752 lung imaging 752 modeling techniques 502-6 myocardial perfusion studies 530–1 neuroreceptor imaging 583–4 neurotransmitters dynamic interactions 603–19 mapping 604 nitrogen-13 labeled ammonia 122, 127

octreotide labeling 650–1 physiological process probing 43 psychostimulants 560–72 radiation labeling 232 radioisotopes 42–53 radioligands 604-5

INDEX

842 positron emission tomography (PET) (contd) radiometals 698 radionuclides 76 receptor study compounds 159–64 region of interest (ROI) 502, 505, 516, 517, 519 serotonin system 590 short-lived positron emitting radionuclides 230–1 tandem cascade accelerator 79 see also FDG-PET; radiotracers, PET; single photon emission computed tomography (SPECT) positron emitters, short-lived 231 post-glomerular capillaries 808 potassium-38 27 potassium bifluoride 311 potassium carbonate 256, 258 potassium iodate 428 potassium/Kryptofix system 197, 198 potentially lethal damage (PLD) repair 770 power deposition 5–6 practolol, carbon-11 labeling 541 pramiprexole 160 prazosin, carbon-11 labeled 543 presenilin genes 488 procaterol, carbon-11 labeled 247 processor-controlled automated devices 142 product burn-up 93 progesterone, carbon-11 labeled 157, 167 progesterone 16a,17a-dioxalanes 7378 progesterone receptors 346, 737 progestin receptor 347, 719 progestins fluorine-substituted 727, 729 iodine substitution 722, 723 technetium-labeled pendant radiopharmaceuticals 732–3 proline, fluorine-18 labeled 213 promethium-149 targeted radiotherapy 644 propanethiol, carbon-11 labeled 246 propenic acid derivatives 148 propionic acid, fluorine-18 labeled 262 proportional integral derivative (PID) control algorithm 291 propranolol (3-adrenergic receptor binding 583 carbon-11 labeled 541 propyl iodide, carbon-11 labeled 243, 244 propyl ketene, carbon-11 labeled 148 propylnorapomorphine 160 prostaglandins, carbon-11 labeled 155 ProstaScint, indium-Ill labeled 365, 377, 382–4, 412, 702–3 skip metastases detection 384 prostate cancer androgen receptor imaging 736–7 androgens 715 detection 377

I8 FDG imaging 757 neurotensin receptors 666 palladium-103 38 ProstaScint 383–4 recurrence diagnosis 365, 383 steroid receptors 346 prostate-specific membrane antigen (PSMA) 383, 703 proteins amino acids reactivity 689 copper labeled 412–14 lysine residues 430 metabolism 798 radioiodination 429–30 protons 91 acceleration 73, 79 reactions 76, 77 protraction effects 771 PSC833 348, 349 psychiatric illness 569 psychostimulants, PET studies 560–72 pulmonary function 751–62 see also lung(s) pulmonary nodules, solitary 631 pulmonary ventilation 90 putamen dopamine levels in Parkinson's disease 586 dopamine secreting neurons 585 putrescine, nitrogen-13 labeled 125 N-pyridoxyl-5-methyltryptophan, technetium-99m labeled 802 pyridoxylidene glutamate, technetium99m labeled 802 pyridoxyline glutamate complex 332 pyrimidine deoxyribose nucleosides, radioiodinated 777 pyrophosphates 333 pyruvate 151

Q value 2-3, 91 Q12 530 quartz ampoules 101 quinoxaline, carbon-11 labeled 158 3-R-quinuclidinyl 4-S-iodobenzilate (RS IQNB) 487–8, 491 stereomers 494–5 3-quinuclidinyl benzilate (QNB) 162, 491, 544 derivatives 454 hydrogen-3 labeled 487 stereomers 494–5 5-I-R91150, iodine-23 labeled 592 raclopride Bmax/Kd' 512 carbon-11 labeled 159, 760, 297, 516–17, 519, 520, 559 cocaine abusers 568 dopaminergic activity modulation 607–8, 609–15 endogenous dopamine competition 605 neurotransmitter studies 606 schizophrenia 587 vigabatrin effects on binding 618

hydrogen-3 labeled 506 nonspecific binding 504 receptor binding 506 radiation detectors 293 labeling 232 quality 771–2 survival curves 768–9 see also irradiation radio gas chromatography 132 radio-HPLC 132 radioactive decay 92 radioactive ions, accelerated 232 radioactivity concentration in plasma/tissue 502 sensors 292-3 radiofluorination, nucleophilic aromatic 726 radiofrequency (RF) oscillator 73 radiohalogens 11–24 radioimmunotherapy astatine-211 695–6 copper-labeled monoclonal antibodies 699 lutetium-177g 108 pretargeted 700–1,705 radionuclide selection 689 yttrium-90 labeled monoclonal antibodies 700–1 radioiodide 428 radioiodine 767 radioisotopes diagnostic 44 production 87–8 proton energies 76 target systems 73 therapeutic 44 see also radionuclides radiolabeled antisense oligodeoxynucleotide (RASON) 469, 478, 479 radioligands binding characteristics 718 brain receptor binding 582 clearance 718 lipophilicity 718 neuropeptides 755 receptor binding 581 receptor trapping 718 serotonin transporter imaging 5912 specific radioactivity 582 target specific 488-96 sensitivity measurement 495–6 radiolysis 42, 47 radionuclides 11–54, 87 accelerator produced 71–81 alpha particle emitters 774–5, 781–3 Auger electron emitters 775–6 availability 739 beta particle emitters 772–4. 778–80, 781 cancer therapy 767-87 experimental 778-86 cellular responses 768–70 choice 772-7 chromatography 102–3

INDEX clinical application 685 copper 401–16 cyclotron-produced 7–70, 71–3, 76– 81 distillation 103 distribution centers 81 dose distribution 773 fractional saturation 717 fractionation effects 771 generator systems 88 half-life 104, 776–7 halogens 11–24 heterogeneity of response 771 impurities 4, 96–7, 107 iodine 423–34 ligand choice 777 metallic 689 metals 29–42, 698–704 availability 698 cost 698 properties 698 molecular lesions 767-8 monoclonal antibody conjugation 779-80 non-metals 24-7 nuclear medicine importance 96, 98 particles 76 PET 76 positron-emitting 42-53 precursors 42 specific activity 43–4 targetry 42 precipitation 103 protraction effects 771 radio biologic effects 767-72 reactor-produced 1, 87–112 chemical processing 102–4 nuclear reactions 88–100 targetry 100–2 relative biologic effectiveness 784, 785 selection 689-90 shielded facilities 103 solvent extraction 102 specific activity 103–4, 717 target properties 778 targeting principles of therapy 772-8 targets 77–8 tissue responses 770–1 vector choice 777 radiopharmaceuticals biodistribution kinetics 796 bromine 448–57 carrier effects on time course 796 copper 401–16 design 718-20 membrane receptor structure 719– 20 receptor-binding 720 effectiveness 771 gallium labeled 369-73 gamma scintigraphy 698 heart studies 529–45 hepatocyte extraction 798 indium labeled 378-92 integrated design 720

843 iodine-containing 426 Kupffer cell extraction 798 liver function study 795–807 mathematical model for receptorspecific 489 pendant/conjugate design 720 radionuclide labeling 769 receptor-binding carbon-11 labeling 729–31 radiometal labeling 731–5 receptor-specific 489, 716 renal function study 795–6, 807–14 renal pathway 810 single dose 88 specific activity 778 structure-distribution relationship (SDR) 490–1 uptake 717 radiophosphorus oxides 777 radioresistance of cells 770 radiotherapy metabolic 426 targeted 644 see also radioimmunotherapy radiotracers biliary excretion 801–3 biochemical variables 796 physiologic variables 796 renal 810,5/2 radiotracers, PET 1, 45, 159–68, 229– 31 automated systems 283-98 application of radiopharmaceuticals 296–7 design/development 285 diagnostics sensor data 293–4 documentation sensor data 2934 feedback control 290 fuzzy logic 291, 294 liquid transfers 291, 292–3 loop method 297 modern synthesizers 294–6 robot-controlled 287, 289, 295–7 solid phase extraction techniques 298 solvent evaporation 291-2 troubleshooting capabilities 293–4 unit operations real-time control 290–3 variable threshold 291 binding/distibution models 502-6 concentration 511 distribution volume 605–6 flow 504 labeled precursor production 231-2 myocardial fatty acid metabolism 532–3, 534 noncompetitive 605–6 physiological function targeting 604 radionuclide incorporation 43 stable species 44 synthesis 159–68 automated systems 283-98 transfer from plasma to irreversible compartment 513

transport 504 radium-224/bismuth-212 generator 704 rare earth colloids 777 reaction probability 91-2 receiver operating characteristic (ROC) curve 806–7 receptor(s) availability for irreversibly binding ligands 508 measures 506–8 binding sites 502 tracer distribution/binding models 503-5 variations 515 binding potential 506, 507, 508, 517– 18

reversible ligands 508 brain 581, 582 compounds for PET study 159–64 concentration for target compound/ maximal bound to free ratio (B/F ratio) 489 free concentration (Bmax') 489, 502, 505, 512, 605–6 identification 583 new 739 imaging 335–7 multiple affinity states 506 noncompetitive ligands 606–7 occupancy 505 sigma 715–16, 720, 723–4 carbon-11 labeling 731 fluorine-substituted ligands 727–8, 730 imaging 738 technetium-labeled 734 tissue concentrations 717 trapping 718 see also nuclear hormone receptors; tumors, receptor imaging receptor-specific agents 335-7 receptor-ligand binding 506 flow limited 515 receptor-ligand dissociation constant 503 receptor-ligand equilibrium dissociation (Kd) 489, 502, 512 radiotracers 605–6 receptor-ligand interactions 487 recoil energy 42 red blood cells, technetium-labeled 324 redistribution of cells 770 relative biologic effectiveness 784, 785 remote-controlled automated devices 142 renal agents 333-5 see also kidneys renal blood flow 811 renal cortical-retention ratio 813 renal excretion index 813–14 renal function evaluation 810-14 measurement 807-10

844 renal function (contd) radiopharmaceutical pathway 810 radiopharmaceuticals for study 795– 6, 807–14 renal imaging iodine-131-Diodrast 426 stannous chloride instant kit 333 technetium-99m-DTPA 324 technetium-99m-glucoheptonate 325 renal perfusion 811 renal plasma flow 807-8 effective 811–12, 814 renal scintigraphy, quantitative 812–13 renal transplant rejection 814 renal tubules acute necrosis 813–14 18 FDG 808 reabsorption 808–10 secretion 808-10 renin 807 renovascular hypertension 813 repair, cellular 770 reporter genes 470 imaging 477-8 reporter probes 470 reticuloendothelial system 797, 800 functional capacity 800–1 imaging 331–3 reward deficiency hypothesis 571 RGD sequence 338, 339, 341 integrins 670, 671-3 tyrosine-containing analogs 672 rhenium 324 bifunctional chelators 699, 704 estrogen receptor integrated ligands 733–4 a-melanocyte-stimulating hormone receptor 338 phosphonate labeling 777 rhenium-186 92–3 monoclonal antibody labeling 703–4 tungsten-188 generation 109,110–11 rhenium-188 antigenic target 686 monoclonal antibody labeling 703–4 rheumatoid arthritis 339 somatostatin receptors 645 rhodium-103 38 ribonucleotide reductase (RR) 371 ring-closure reactions 147, 156–7, 158– 9 formaldehyde preparations 148 risperidone 594 ritalin see methylphenidate Ro 15-1788, carbon-11 labeled 297 Ro 15–5528 287, 289, 295–6 robots see automated systems, robotcontrolled Rolipram, carbon-11 labeled 297 Rose Bengal, iodine-131 labeled 332, 801-2 rotenoids, carbon-11 labeled 164 roxithromycine, carbon- II labeled 168 RP593 338 RU486 346 rubidium 36

INDEX rubidium-81 /krypton-Sim generator 752 rubidium-82 530 38S1 monoclonal antibody, bromine-76 labeled 457 salmon calcitonin 665–6 salt, molten 21-2 samarium, phosphonate labeling 777 Sandmeyer reaction 448 SarCNU, carbon-11 labeled 168 sarcoidosis, gaIlium-67 citrate 370 sarcoma staging with FDG-PET 633 saturation factor 92-3 scandium 28 Schering 23390, carbon-11 labeled 297, 517 Schiffbase 348 ligand 329 schizophrenia 571, 587 cholinergic/dopaminergic interaction model 615 cholinergic system 615 DAT as candidate gene 587 dopamine receptors 584 imaging 585 GABA action 610 GABAergic modulation of dopamine 612-15 5-HTM receptors 591, 594 muscarinic receptors 615 raclopride carbon-11 labeled binding 605

vigabatrin treatment 615 scopolamine 515 second messenger system, signal transduction tracers 164 secretin, nitrogen-13 labeled 127 selective serotonin reuptake inhibitors (SSRIs) 590, 609 depression in Parkinson's disease 592 selenium 446 carbonylation 156, 157 elemental 16 selenium-72 25 selenium-73 25–6 selenium-75 25–6, 441 selenium-77 445 selenomethionine, carbon-11 labeled 165

self-shielding 93 semiconductor technology 286 semotiadil, carbon-11 labeled 163 separation of mass-to-charge ration (m/z) 448 sepharose, cyanogen bromide activated 123 serine, carbon-11 labeled 153 serotonergic ligands 160–1 serotonergic system 589–94 Alzheimer's disease 593 depression 593-4 Parkinson's disease 592–3 serotonin 589 acetylcholine modulation 617 autoreceptors 589

carbon-11 labeled 762 dopamine modulation 609–10 interactions in extra-pyramidal motor system 607 lungs 762 molecular mechanisms of release 589 platelet uptake 593 terminal loss 591 see 5-HT receptors serotonin transporter 589, 590 allelic variation 594 Alzheimer's disease 593 cocaine binding 560 depression 594 mRNA 590 promoter genotyping 594 radioligands 5 9 1 2 transmembrane domains 586 serotonin transporter (SERT) 160, 342, 343 bromine-76 labeled 454 sestamibi 328, 529–30 setoperone, fluorine-18 labeling 594 sex hormone-binding globulin 735 sialic acid 803 sigma receptors 345–6 silica beads, porous derivatized 123 silver 38 simplified reference tissue method (SRTM) 517–18 single-chain Fv fragment (ScFv) 687, 688 single photon emission computed tomography (SPECT) bromine isotopes 441 imaging data quantification 502-21 lung imaging 752 myocardial viability 531 serotonin system 590 technetium-99m-GSA 806 tracers for myocardial fatty acid metabolism 534–5 Sjogren's syndrome, gallium-67 citrate 370 skeletal imaging 333 technetium-99m-MDP 325 technetium-99m-pyrophosphate 324 SKF 82957, carbon-11 labeled 297 skip metastases detection 384 smoking, brain changes 568-71 sodium chloride 27 sodium cyanoborohydride 267 sodium iodide 429. 430. 431 carrier-free 429 sodium pertechnate 324 solid phase extraction techniques 298 solid phase peptide synthesis (SPPS) 284 solid targets 78 somatostatin 387, 414, 645–6 amino acids 645–6 analogs 386, 414, 415 iodine-123 labeled analogs 646 somatostatin receptors (SSTR) 387, 388,414 binding 645-6

INDEX indium-Ill DTPA octreotide 648 inflammatory conditions 645 reporter gene imaging 477-8 subtypes 646 sonication 142 space of Disse 800, 801 spallation process 91 specific activity 43–4 molybdenum-99 105-6 specific binding equation 487 spiperone 446 analogs 455, 584 derivatives 208, 266 fluorine-18 labeled 517 receptor binding 506 see aho N-methylspiperone spiroperidols, fluorine-18 labeled 259, 260 spleen agents 332 hSSTr2 reporter gene 478 stannous chloride, instant kit 333 steroid receptors 346–7 steroids carbon-11 labeled 167–8 catabolism 798 fluorine-18 labeled 207–8 Stille couplings 154 stopping power 5–6 streptavidin 780, 781 streptozotocin, nitrogen-13 labeled 127 striatum cholinergic cells 616 dopamine transporter protein distribution 586 serotonergic enervation 617 strontium-82 93 strontium-86 36 structure-distribution relationship (SDR) 490-1 subiethal damage (SLD) repair 770 substance abuse DAT as candidate gene 587 GABAergic modulation of dopamine 611-12 substance P 658–9, 751 analogs 660 substance P receptors 658–9 substantia nigra dopamine D4 receptors 587 dopaminergic projections 611 neurons degeneration 582 dopamine secretion 585 W-succinimidy! 1 -{(4'-fluorobenzylamino)]suberate, fluorine-18 labeled 697 //-succinimidyl 3-astatobenzoate (SAB), astatine-211 labeled 696 A'-succinimidyl 3-hydroxy-4-iodobenzoate 427 A'-succinimidyl 3-(tri-n-butyl)stannylbenzoate 691 A'-succinimidyl 4-fluorobenzoate, fluorine-18 labeled 262, 697 N-mcd aimidyl 4-(fluoromethyl)benzoate, fluorine-!8 labeled 697-8

845 Af-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) 693, 694 jV-succinimidyl 8-(4-fluorobenzyl)amino substrates, fluorine-18 labeled 262 Af-succinimidyl bromobenzoate (BrNHS), bromine-76 labeled 456 jV-succinimidyl iodobenzoates 691–2 Af-succinimidyl para-iodobenzoate 427 sulfation pathway 798 sulfonamides 147 fluorine-18 labeling 255 sulfur colloid, technetium-99m labeled 799, 800–1 sulfuric acid 317 superoxide dismutase (SOD) mimics 405 survival 768-9 Suzuki couplings 154 sympathetic nerve imaging, myocardial 537–40 oc-synuclein 588 systemic lupus erythematosus (SLE), gallium-67 citrate 370 Szilard-Chaimers process 97 tachykinin receptor subtypes 658 tachykinins 658 TAG-72 tumor-associated antigen 385, 702 tamoxifen 735, 737 carbon-11 labeled 168 tantalum-178 40–1 tardive dyskinesia, GAB A action 610 target material 6 availability 101 chemical form 100-1 chemical stability 101 encapsulation 101 enriched 104 hydraulic tube facilities 102 physical form 100–1 pneumatic tube facilities 101-2 purity 101 thermal properties 101 transport systems 101 target specific uptake mechanisms 48796 radioactive analog stereomers 494– 5 radioactive derivative preparation 492–4 radioligands 487, 488–96 teboroxime 530 Technegas 754 technetium acetanilidoiminodiacetate complex 332 amyloid plaque 344–5 antibody fragment labeling 336 autologous erythrocytes 332 azo dye labeling 344, 345 BATO-2MP complex 327 BATO complex 326–7, 329–30 bifunctional chelators 699 bone marrow agents 331, 333

boronic acid adducts of dioximes 326, 327 brain agents 325-7 ciprofloxacin labeling 339 Cl2(dmpe)2 complexes 329 complex interactions with enzymes/ cellular proteins 335 DMSA 334 DTPA 333, 334 estradiol labeling 347 estrogen receptor integrated ligands 734 glucoheptonate complex 333, 334 heart agents 327-30 hepatobiliary agents 331, 332–3 hypoxia agents 330–1 infection imaging agents 339 inflammation imaging agents 339 lung agents 331-2 lymph node agents 331 macroaggregated albumin 331–2 a-melanocyte-stimulating hormone (a-MSH) receptor 338 metal binding unit 732 methylene diphosphonate complex 333 neuroreceptor agents 341–4, 345–7 NOET 330 organic molecule complexes 336–7 penicillamine complex 332 peptide complexes 336 pyridoxyline glutamate complex 332 radiopharmaceuticals 323–49 applications 324–49 chemistry 324 exchange labeling 324 transmetalation 324 receptor-binding radiopharmaceutical labeling 731–5 receptor-specific agents 335–7 red blood cell labeling 324 Schiff base ligand 329 sigma receptor labeling 345–6 steroid receptor labeling 346–7 thrombus agents 339–43 transporter agents 341–4, 345–7 technetium(I) complexes 328 isonitrile complexes 328–9 technetium(III) 327 technetium(V) complexes 325, 326 technetium-94m 37-8 receptor-binding radiopharmaceutical labeling 732 technetium-99, pertechnate ion 323 technetium-99m 88, 90 annexin V 336 antimony trisulfide colloid 331 BAT-EN6 345 brain agents 325–7 calcitonin analog labeling 665–6 calcium phytate 331 carbon particle labeling 754 DAT 342 diagnostic use 104

INDEX

846 technetium-99m (contd) diethylene triamine pentaacetic acid labeling 754 N-(2,6-dimethylphenyolcarbamoylmethyl)-iminodiacetic acid (HIDA) labeling 325, 802–3 disofenin labeling 803 DTPA labeling 324, 808, 812, 813 ECD 335 galactosyl-neoglycoalbumin labeling 332, 333, 804–5, 806–7 generation 698 glucoheptonate 325 GP Ilb/IIIa receptor antagonist labeling 339–41 GSA 804–6 half-life 323 HL-91 labeling 331, 537 HMPAO 325, 327 infection/inflammation site imaging 339 5-HTIA and 5-HT2A receptor binding 343–4 human serum galactosyl albumin (GSA) 332–3 iminodiacetic acid labeling 802–3 lipophilic complexes 529–30 macroaggregated albumin labeling 754 MDP 325 mebrofenin labeling 803 mercaptoacetyltriglycine (MAGs) labeling 334, 335, 704, 809, 811 microaggregated albumin 331 mode of decay 323 monoclonal antibody labeling 7012 nitroimidazole derivatives 330 nofetumomab merpentan 701 nonpenetrating radiation 323 octreotide labeling 649 P2 338 PAC1.1 monoclonal antibody labeling 341 pertechnate ion 323 phytate labeling 799 PNAO 330–1 polyphosphate 333 production 104–6 propyleneamine oxime (PNAO) complexes 325 N-pyridoxyl-5-methyltryptophan 802 pyridoxylidene glutamate labeling 802 pyrophosphate 324 Q complexes 329, 348 receptor-binding radiopharmaceutical labeling 731 RP593 labeling 338 RU486 346 separation of high-specific-activity samples 323 sulfur colloid 331 labeling 799, 800–1 teboroxime 329–30

technepine 342–3 tetrofosmin 329, 348 tin colloids 331 TP3654 labeling 658 TRODAT 342 TRODAT-1 labeling 518 tropane labeling 342 tuftsin 339 vasoactive intestinal peptide labeling 657 white blood cells 392 technetium-99m(IXMIBI)6 + 329, 335 transport substrate role 348 tumor accumulation 349 technetiumvO complexes of MPP 343, 344 tellurium 20, 21, 22, 23, 25 astatine-211 colloid 783 tellurium-124 18, 20, 23 tellurium oxide 21, 22, 23 tellurium-gold alloy 21 terbium-161 targeted radiotherapy 644 TETA copper labeled bifunctional chelators 404, 405, 412–13 octreotide conjugation 414, 415 tetraalkylammonium salts 197, 198, 257 tetraazacyclodecanetetraacetic acid see DOTA tetrabutylammonium 316 tetrabutylammonium bichromate 241 tetrabutylammonium fluoride 245 tetrachloromethane 447 tetrakis(triphenylphosphine)palladium (0) 237, 238 tetralin derivatives 160 tetraphenyl tin 97 tetrofosmin 530 thallates, organic 433 thallium-201 41–2, 327 thallium-208 704 thallium(III) trifluoroacetate (TTFA) 433 thermal neutrons 89, 92, 93 fission 90 flux 95 thimble mechanisms 102 thiocyanates, carbon-11 labeled 149 thionyl bromide 265 thionyl chloride 265 thiophene derivatives 433 metallation 432–3 bis(thiosemicarbazone) derivatives 636 thiosemicarbazones 404, 636 threshold energy 91 thrombocytopenia 382 thrombus agents 339–41 thrombus detection 449 thymidine analogs 442, 450–2 carbon-11 labeled 168, 242, 635 thymidine kinase 635 see also herpes simplex virus thymidine kinase (HSVl-tk)

thyroid cancer 90 cholecystokinin B (CCK-B)/gastrin receptor 667 indium-Ill DTPA octreotide 648–9 iodine transporter 777 radioiodine therapy 767 substance P receptors 659 thyroid gland hyperactive 90 radioiodine 424 tin 87 colloids 331 tin-110 39 tin-117m 97 radionuclide impurities 107 reactor production 106–7 tin-119m 106–7 tissue concentration 511 tissue responses to adionuclides 770–1 titanium 28 titanium-48 28 titanium (III) chloride 123 titanium (III) hydroxide 123 tobacco smoke, brain changes 568–71 toremifene 168 Tourette syndrome DAT as candidate gene 587 dopamine system 589 TP-1 689 TP-3 689 TP3654 657-8 technetium-99m labeled 658 transferrin 365, 367 binding 367 carbon-II labeled 168 gallium-67 binding 370, 371 gallium-67 uptake in tumor cells 370–1 gallium-68 binding 373 indium-Ill binding 376 indium-113m binding 762 indium binding 374–5 receptors 370, 371 saturation with iron 371–2 transgene expression imaging 469–70 transgenic technology 739 transglutaminase 127 transition metals 153–7 transjugular intrahepatic portosystemic shunt (TIPS) 805–6 transmetalation 324 transplant rejection, apoptosis detection 336 transport constants 504, 520 transporter agents 341–4, 345–7 3,4,6-tri-O-acetylglucal, electrophilic fluorination 311 tributylphosphonium 316 tricarbonylchromium complex 237 tricarbonyltechnetium complex 652 tricyclic antidepressants 163 inflates 261 16p-trifloxy-estrone 3-triflate 725 trifluoracetonitrile. carbon-11 labeled 149 trifluoroacetic acid 433

INDEX bis-(trifluoroacetoxy)thallium 433 trifluoromethyl hypofluorite 251, 311 trifluoromethylsulfonates 261 see also triflate compounds triisopropylsilyl 4-(dimethylboro) butanote 125 trimethylsilyl chloride 147 /V-trimethylsilyipyridinium triflate 253 triphenylarsonium methylide 147 triphenylphosphine 247, 447 trisuccin 661 tritium see hydrogen-3 TROD AT, technetium-99ni labeled 342 TRODAT-1, technetium-99m labeled 518 tropanes fluorine-18 labeled 209–10 techrtetium-99m labeled 342 tropany! benzilate, carbon-31 labeled 515, 606 trovafloxacin, fluorine-18 labeled 210, 211 t-tryptophan 151 tryptophan, fluorine-18 labeled 203 tuftsin 339 tumor(s) accessibility 778 amino acid metabolism 245, 634–5 angiogenesis markers 669-70 RGD sequences 671–2 binding sites 778 bombesin uptake 662 calcitonin receptors 664 chemoresistance 636 cholecystokinin uptake 669 copper-62-ATSM 412 depreotide uptake 655 detection/staging 392-3 I8 FDG uptake 311, 630 glucose uptake 630 hypoxia imaging 330, 331, 636 imaging 629–36 radiolabeled peptides 643–75 i n d i u m - I l l DTPA octreotide 648–9 imaging 657 i n d i u m - I l l labeled OctreoScan 388, 389 integrin uptake 671, 672, 673 microscopic organization 778 microsomal bioreductive enzymes 412 monoclonal antibody accumulation 686

multidrug resistance 335, 347-9 oxygenation 778 proliferation 635 protein synthesis 634–5 radioactivity targeting approaches 780, 781 radiotmmunodetection 426 radiolabeled antibodies for imaging/ therapy 685–705 radiolabeled antigens 426–7 radioresistance 636

receptor imaging 715–39, 716 agent synthesis 718–20 bromine radioisotopes 721–4 carbon-11 labeling 729–31 fluorine-18 labeling 724–8, 729 gallium radionuclides 734–5 iodine radioisotopes 721–4 medical applications 735–8 radiochemistry for agent synthesis 721-31 radiometal labeling 731–5 target-to-background contrast 718 uptake over background 718 uptake process 717 receptor-mediated uptake 717, 718 receptor structure 718–20 receptor tissue concentrations 717 soft tissue 370 somatostatin receptors 645, 656 targeting 414 steroid receptor assays 716 transferrin receptors 370, 371 vascular permeability 778 vascularity 778 vasoactive intestinal peptide iodine-123 imaging 657 uptake 656 vasoactive intestinal peptide receptors 656, 658 tumor agents 337-9 tumor-associated antigens 427, 777 tumor cells bombesin receptors 662 glucose metabolism 630 octreotide, indium-111 DTPA internalizing 647 tumor-seeking agents 30 tungsten, metallic 110 tungsten-188 104 nuclear reactor production 109–10 specific activity 111 tungsten-188/rhenium-188 generator system 110–11 two shoot method 46 tyramine, carbon-11 labeled 167 tyramine-cellobiose 694 radioiodinated 693 tyrosinase 470 tyrosine 431 fluorine-18 labeled 203–4, 205 monoclonal antibodies V domain 688-9 octreotide analog 646 t-tyrosine 151 tyrosine kinase inhibitors 164 [tyrosine3] octreotate (Y3-TATE) 6512

iodine-125 glycated 653 [tyrosine3] octreotide (TOC) 651 iodine-125 glycated 652-3 turner uptake 653 ubiquitin cascade of protein degradation 588 ubiquitin-protein ligase 588

UCH-L1 enzyme 588 ulcerated colitis, somatostatin receptors 645 ultraviolet light detection 448 umpolung 149 unit operations design 288-9 uracil analogs 471, 472, 476–7 uranium-235 enriched 94 fission 93–4, 105 neutron-induced 99-100 urea carbon-11 labeled 148, 242, 246–7 formation 146, 157 nitrogen-13 labeled 126 ureteral obstruction 814 uterine cancer staging with FDG-PET 633 valium 583 vanadium-48 28 vapreotide 653–4 vasa recta 809–10 vasoactive intestinal peptide (VIP) 655-8, 751 active site 656 analogs 657-8 biologic activity 656 fluorine-18 labeled 658 half-life in blood 657 iodine-123 labeled 656 nitrogen-13 labeled 127 receptors 337, 655, 658 technetium-99m labeled 657 tumor uptake 656 ventilation 751 gases 752–4 particles in measurement 754–5 ventilation/perfusion ratio 752–3 ventral pallidum 618 ventral striatal region 618 ventral tegrnental area 611 vigabatrin 610, 611–12, 613, 614 endogenous GABA activity 618 schizophrenic psychosis 615 vinylbromide 238 vinylstannanes 432 VIPomas 656 viral infection imaging agents 471 visual cortex, FDG accumulation 584 vitronectin receptor 338 W-178 40 Wallach reaction 432 water carbon-11 labeled 241 frozen 123 nitrogen-13 production 754 oxygen-15 labeled 53, 130, 131, 530–1 renal perfusion 811 oxygen-18 enriched 47, 196. 197, 250, 255, 314 radiolysis products 47 target 47–8, 52 water for injection, labeling 52 wavelet transform 520

848 WAY 100634 160, 161 WAY 100635 343 binding potential 590 carbon-11 labeled 517-18, 590, 593 white blood cells indium-111 labeled 377, 378–80 technetium-99m labeled 392 WIN 35428 hydrogen-3 labeled 560 Parkinson's disease 588 Wittig reaction 148, 155 phosphonium salts 247–8 wolfram see tungsten x-rays 769 xanomeline 162 xenon 22 xenon-123 18, 20 beta decay 428 decay 428

INDEX neutrino emission 428 xenon-124 22 xenon-125 98–9 xenon-127 91, 752 xenon-133 90, 100, 752, 753 xenon-135 94 xenon difluoride, fluorine-18 labeling 252-3 Y3-TATE somatostatin analog 415 ytterbium-176 109 yttrium-86 36 yttrium-89 36 yttrium-90 393 antigenic target 686 biotin labeling 780, 757 bone pain palliation 36 ' linear energy transfer 772 monoclonal antibody labeling 689– 90, 700-1, 780

short peptide labeling 777 targeted radiotherapy 644 yttrium-90-DOTA-octreotide 389 yttrium-90-Zevalin 393 yttrium colloids 777 Zevalin, indium-111 labeled 393 zinc 24, 34 catalytic reduction 235 zanc-62 32, 35 copper-62 production 403 zinc-62/copper-62 generator 403, 409, 410

zinc-63 35–6 zinc-64(n,p)copper-64 reaction 402 zinc-67, copper-67 production 112 zinc-68{p,2n)gallium-67 reaction 402 zirconium-89 36-7 zirconium foil 37